Production of Polyketides

ABSTRACT

Recombinant host cells of the suborder Cystobacterineae containing recombinant expression vectors that encode heterologous PKS genes can produce polyketides synthesized by the PKS enzymes encoded on those vectors at high levels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application Ser. No.09/443,501, filed 19 Nov. 1999; PCT patent application US99/27438, filed19 Nov. 1999; and U.S. provisional application Ser. Nos. 60/130,560,filed 22 Apr. 1999; 60/122,620, filed 3 Mar. 1999; 60/119,386, filed 10Feb. 1999; and 60/109,401, filed 20 Nov. 1998, each of which isincorporated herein by reference.

REFERENCE TO GOVERNMENT FUNDING

This invention was supported in part by SBIR grant 1R43-CA79228-01. TheU.S. government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention provides recombinant methods and materials forproducing polyketides in recombinant host cells. The recombinant hostcells are from the suborder Cystobacterineae, preferably from the generaMyxococcus and Stigmatella that have been transformed with recombinantDNA expression vectors of the invention that encode modular or iterativepolyketide synthase (PKS) genes. The recombinant host cells produceknown and novel polyketides, including but not limited to epothilone andepothilone derivatives. The invention relates to the fields ofagriculture, chemistry, medicinal chemistry, medicine, molecularbiology, and pharmacology.

BACKGROUND OF THE INVENTION

Polyketides constitute a class of structurally diverse compoundssynthesized, at least in part, from two carbon unit building blockcompounds through a series of Claisen type condensations and subsequentmodifications. Polyketides include antibiotics such as tetracycline anderythromycin, anticancer agents such as epothilone and daunomycin, andimmunosuppressants such as FK506 and rapamycin. Polyketides occurnaturally in many types of organisms, including fungi and mycelialbacteria. Polyketides are synthesized in vivo by polyketides synthaseenzymes commonly referred to as PKS enzymes. Two major types of PKS areknown that differ in their structure and the manner in which theysynthesize polyketides. These two types are commonly referred to as TypeI or modular and Type II or iterative (aromatic) PKS enzymes.

The present invention provides methods and recombinant expressionvectors and host cells for the production of modular or iterative PKSenzymes and the polyketides produced by those enzymes. Modular PKSenzymes are typically multi-protein complexes in which each proteincontains multiple active sites, each of which is used only once duringcarbon chain assembly and modification. Iterative PKS enzymes aretypically multi-protein complexes in which each protein contains onlyone or at most two active sites, each of which is used multiple timesduring carbon chain assembly and modification. As described in moredetail below, a large number of the genes for both modular and aromaticPKS enzymes have been cloned.

Modular PKS genes are composed of coding sequences organized to encode aloading module, a number of extender modules, and a releasing domain. Asdescribed more fully below, each of these domains and modulescorresponds to a polypeptide with one or more specific functions.Generally, the loading module is responsible for binding the firstbuilding block used to synthesize the polyketide and transferring it tothe first extender module. The building blocks used to form complexpolyketides are typically acylthioesters, most commonly acetyl,propionyl, malonyl, methylmalonyl, hydroxymalonyl, methoxymalonyl, andethylmalonyl CoA. Other building blocks include amino acid-likeacylthioesters. PKSs catalyze the biosynthesis of polyketides throughrepeated, decarboxylative Claisen condensations between theacylthioester building blocks. Each module is responsible for binding abuilding block, performing one or more functions on that building block,and transferring the resulting compound to the next module. The nextmodule, in turn, is responsible for attaching the next building blockand transferring the growing compound to the next module until synthesisis complete. At that point, the releasing domain, often an enzymaticthioesterase (TE) activity, cleaves the polyketide from the PKS.

The polyketide known as 6-deoxyerythronolide B (6-dEB) is synthesized bya prototypical modular PKS enzyme. The genes, known as eryAI, eryAII,and eryAIII, that code for the multi-subunit protein known asdeoxyerythronolide B synthase or DEBS (each subunit is known as DEBS1,DEBS2, or DEBS3) that synthesizes 6-dEB are described in U.S. Pat. Nos.5,712,146 and 5,824,513, incorporated herein by reference.

The loading module of the DEBS PKS consists of an acyltransferase (AT)and an acyl carrier protein (ACP). The AT of the DEBS loading modulerecognizes propionyl CoA (other loading module ATs can recognize otheracyl-CoAs, such as acetyl, malonyl, methylmalonyl, or butyryl CoA) andtransfers it as a thioester to the ACP of the loading module.Concurrently, the AT on each of the six extender modules of DEBSrecognizes a methylmalonyl CoA (other extender module ATs can recognizeother CoAs, such as malonyl or alpha-substituted malonyl CoAs, i.e.,malonyl, ethylmalonyl, and 2-hydroxymalonyl CoA) and transfers it to theACP of that module to form a thioester. Once DEBS is primed with acyl-and methylmalonyl-ACPs, the acyl group of the loading module migrates toform a thioester (trans-esterification) at the KS of the first extendermodule; at this stage, module one possesses an acyl-KS adjacent to amethylmalonyl ACP. The acyl group derived from the DEBS loading moduleis then covalently attached to the alpha-carbon of the extender group toform a carbon-carbon bond, driven by concomitant decarboxylation, andgenerating a new acyl-ACP that has a backbone two carbons longer thanthe loading unit (elongation or extension). The growing polyketide chainis transferred from the ACP to the KS of the next module of DEBS, andthe process continues.

The polyketide chain, growing by two carbons for each module of DEBS, issequentially passed as a covalently bound thioester from module tomodule, in an assembly line-like process. The carbon chain produced bythis process alone would possess a ketone at every other carbon atom,producing a polyketone, from which the name polyketide arises. Commonly,however, additional enzymatic activities modify the beta keto group ofeach two carbon unit just after it has been added to the growingpolyketide chain but before it is transferred to the next module. Thus,in addition to the minimal module containing KS, AT, and ACP necessaryto form the carbon-carbon bond, modules may contain a ketoreductase (KR)that reduces the keto group to an alcohol. Modules may also contain a KRplus a dehydratase (DH) that dehydrates the alcohol to a double bond.Modules may also contain a KR, a DH, and an enoylreductase (ER) thatconverts the double bond to a saturated single bond using the betacarbon as a methylene function. The DEBS modules include those with onlya KR domain, only an inactive KR domain, and with all three KR, DH, andER domains.

Once a polyketide chain traverses the final module of a PKS, itencounters the releasing domain, typically a thioesterase, found at thecarboxyl end of most modular PKS enzymes. Here, the polyketide iscleaved from the enzyme and, for many but not all polyketides, cyclized.The polyketide can be modified further by tailoring or modificationenzymes; these enzymes add carbohydrate groups or methyl groups, or makeother modifications, i.e., oxidation or reduction, on the polyketidecore molecule. For example, 6-dEB is hydroxylated, methylated, andglycosylated (glycosidated) to yield the well known antibioticerythromycin A in the Saccharopolyspora erythraea cells in which it isproduced naturally.

While the above description applies generally to modular PKS enzymes andspecifically to DEBS, there are a number of variations that exist innature. For example, many PKS enzymes comprise loading modules that,unlike the loading module of DEBS, comprise an “inactive” KS domain thatfunctions as a decarboxylase. This inactive KS is in most instancescalled KS^(Q), where the superscript is the single-letter abbreviationfor the amino acid (glutamine) that is present instead of the activesite cysteine required for ketosynthase activity. The epothilone PKSloading module contains a KS^(Y) domain in which tyrosine has replacedthe cysteine. Moreover, the synthesis of other polyketides begins withstarter units that are unlike those bound by the DEBS or epothiloneloading modules. The enzymes that bind such starter units can include,for example, an AMP ligase such as that employed in the biosynthesis ofFK520, FK506, and rapamycin, a non-ribosomal peptide synthase (NRPS)such as that employed in the biosynthesis of leinamycin, or a solubleCoA ligase.

Other important variations in PKS enzymes relate to the types ofbuilding blocks incorporated as extender units. As for starter units,some PKS enzymes incorporate amino acid like acylthioester buildingblocks using one or more NRPS modules as extender modules. Theepothilone PKS, for example, contains an NRPS module. Another suchvariation is found in the FK506, FK520, and rapamycin PKS enzymes, whichcontain an NRPS that incorporates a pipecolate residue and also servesas the releasing domain of the PKS. Yet another variation relates toadditional activities in an extender module. For example, one module ofthe epothilone PKS contains a methyltransferase (MT) domain, whichincorporates a methyl group into the polyketide.

Recombinant methods for manipulating modular and iterative PKS genesthat take advantage of the organization of those genes and the multipleenzymatic activities they encode are described in U.S. Pat. Nos.5,672,491; 5,712,146; 5,830,750; and 5,843,718; and in PCT patentpublication Nos. 98/49315 and 97/02358, each of which is incorporatedherein by reference. These and other patents describe recombinantexpression vectors for the heterologous production of polyketides aswell as recombinant PKS genes assembled by combining parts of two ormore different PKS genes that produce novel polyketides. To date, suchmethods have been used to produce known or novel polyketides inorganisms such as Streptomyces, which naturally produce polyketides, andE. coli and yeast, which do not naturally produce polyketides (see U.S.Pat. No. 6,033,883, incorporated herein by reference). In the latterhosts, polyketide production is dependent on the heterologous expressionof a phosphopantetheinyl transferase, which activates the ACP domains ofthe PKS (see PCT publication No. 97/13845, incorporated herein byreference).

While such methods are valuable and highly useful, certain polyketidesare expressed only at very low levels or are toxic to the heterologoushost cell employed. As an example, the anticancer agent epothilone wasproduced in Streptomyces by heterologous expression of the epothilonePKS genes (Tang et al., 28 Jan. 2000, Cloning and heterologousexpression of the epothilone gene cluster, Science, 287:640-642, andU.S. patent application Ser. No. 09/443,501, filed 19 Nov. 1999, each ofwhich is incorporated herein by reference). However, the production ofepothilone was only about 50 to 100 μg/L and appeared to have adeleterious effect on the producer cells.

The epothilones were first identified as an antifungal activityextracted from the myxobacterium Sorangium cellulosum (see K. Gerth etal., 1996, J. Antibiotics 49: 560-563 and Germany Patent No. DE 41 38042, each of which is incorporated herein by reference) and later-foundto have activity in a tubulin polymerization assay (see Bollag et al.,1995, Cancer Res. 55:2325-2333, incorporated herein by reference). Theepothilones have since been extensively studied as potential antitumoragents for the treatment of cancer. The chemical structure of theepothilones-produced by Sorangium cellulosum strain So ce 90 wasdescribed in Hofle et al., 1996, Epothilone A and B—novel 16-memberedmacrolides with cytotoxic activity: isolation, crystal structure, andconformation in solution, Angew. Chem. Int. Ed. Engl. 35(13/14):1567-1569, incorporated herein by reference. The strain was found toproduce two epothilone compounds, designated A (R═H) and B (R═CH₃), asshown below, which showed broad cytotoxic activity against eukaryoticcells and noticeable activity and selectivity against breast and colontumor cell lines.

The desoxy counterparts of epothilones A and B, also known asepothilones C(R═H) and D (R═CH₃), are known to be less cytotoxic, andthe structures of these epothilones are shown below.

Two other naturally occurring epothilones have been described. These areepothilones E and F, in which the methyl side chain of the thiazolemoiety of epothilones A and B has been hydroxylated to yield epothilonesE and F, respectively.

Because of the potential for use of the epothilones as anticanceragents, and because of the low levels of epothilone produced by thenative So ce 90 strain, a number of research teams undertook the effortto synthesize the epothilones. This effort has been successful (seeBalog et al., 1996, Total synthesis of (−)-epothilone A, Angew. Chem.Int. Ed. Engl. 35(23/24): 2801-2803; Su et al., 1997, Total synthesis of(−)-epothilone B: an extension of the Suzuki coupling method andinsights into structure-activity relationships of the epothilones,Angew. Chem. Int. Ed. Engl. 36(7): 757-759; Meng et al., 1997, Totalsyntheses of epothilones A and B, JACS 119(42): 10073-10092; and Baloget al., 1998, A novel aldol condensation with 2-methyl-4-pentenal andits application to an improved total synthesis of epothilone B, Angew.Chem. Int. Ed. Engl. 37(19): 2675-2678, each of which is incorporatedherein by reference). Despite the success of these efforts, the chemicalsynthesis of the epothilones is tedious, time-consuming, and expensive.Indeed, the methods have been characterized as impractical for thefull-scale pharmaceutical development of an epothilone.

A number of epothilone derivatives, as well as epothilones A-D, havebeen studied in vitro and in vivo (see Su et al., 1997,Structure-activity relationships of the epothilones and the first invivo comparison with paclitaxel, Angew. Chem. Int. Ed. Engl. 36(19):2093-2096; and Chou et al., August 1998, Desoxyepothilone B: anefficacious microtubule-targeted antitumor agent with a promising invivo profile relative to epothilone B, Proc. Natl. Acad. Sci. USA 95:9642-9647, each of which is incorporated herein by reference).Additional epothilone derivatives and methods for synthesizingepothilones and epothilone derivatives are described in PCT patentpublication Nos. 00/00485, 99/67253, 99/67252, 99/65913, 99/54330,99/54319, 99/54318, 99/43653, 99/43320, 99/42602, 99/40047, 99/27890,99/07692, 99/02514, 99/01124, 98/25929, 98/22461, 98/08849, and97/19086; U.S. Pat. No. 5,969,145; and Germany patent publication No. DE41 38 042, each of which is incorporated herein by reference.

There remains a need for economical means to produce not only thenaturally occurring epothilones but also the derivatives or precursorsthereof, as well as new epothilone derivatives with improved properties.There remains a need for a host cell that produces epothilones orepothilone derivatives that is easier to manipulate and ferment than thenatural producer Sorangium cellulosum yet produces more of the desiredpolyketide product. The present invention meets these by providing hostcells that produce polyketides at high levels and are useful in theproduction of not only epothilones, including new epothilone derivativesdescribed herein, but also other polyketides.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides recombinant host cellsof the suborder Cystobacterineae containing recombinant expressionvectors that encode heterologous PKS genes and produce polyketidessynthesized by the PKS enzymes encoded on those vectors. In a preferredembodiment, the host cells are from the genus Myxococcus or the genusStigmatella. In especially preferred embodiments, the host cells areselected from the group consisting of M. stipitatus, M. fulvus, M.xanthus, M. virescens, S. erects, and S. aurantiaca.

In another embodiment, the present invention provides recombinant DNAvectors capable of chromosomal integration or extrachromosomalreplication in the host cells of the invention. The vectors of theinvention comprise at least a portion of a PKS coding sequence and arecapable of directing expression of a functional PKS enzyme in the hostcells of the invention.

In another embodiment, the present invention provides a method forproducing a polyketide in a host cell of the suborder Cystobacterineae,which polyketide is not naturally produced in said host cell, saidmethod comprising culturing the host cell transformed with a recombinantDNA vector of the invention under conditions such that a PKS geneencoded on the vector is expressed and said polyketide is produced.

In a preferred embodiment, the recombinant host cell of the inventionproduces epothilone or an epothilone derivative. Thus, the presentinvention provides recombinant host cells that produce a desiredepothilone or epothilone derivative. In a preferred embodiment, the hostcell produces the epothilones at equal to or greater than 10 mg/L. Inone embodiment, the invention provides host cells that produce one ormore of the epothilones or epothilone derivatives at higher levels thanproduced in the naturally occurring organisms that produce epothilones.In another embodiment, the invention provides host cells that producemixtures of epothilones that are less complex than the mixtures producedby naturally occurring host cells that produce epothilones.

In an especially preferred embodiment, the host cells of the inventionproduce less complex mixtures of epothilones than do naturally occurringcells that produce epothilones. Naturally occurring cells that produceepothilones typically produce a mixture of epothilones A, B, C, D, E,and F. The table below summarizes the epothilones produced in differentillustrative host cells of the invention.

Cell Type Epothilones Produced Epothilones Not Produced 1 A, B, C, D E,F 2 A, C B, D, E, F 3 B, D A, C, E, F 4 B A, C, D, E, F 5 D A, B, C, E,FThus, the recombinant host cells of the invention also include hostcells that produce only one desired epothilone or epothilone derivative.

In a related preferred embodiment, the invention provides recombinantDNA expression vectors that encode all or a portion of the epothilonePKS. Thus, the present invention provides recombinant DNA expressionvectors that encode the proteins required to produce epothilones A, B,C, and D in the host cells of the invention. The present invention alsoprovides recombinant DNA expression vectors that encode portions ofthese proteins. The present invention also provides recombinant DNAcompounds that encode a hybrid protein, which hybrid protein includesall or a portion of a protein involved in epothilone biosynthesis andall or a portion of a protein involved in the biosynthesis of anotherpolyketide or non-ribosomal-derived peptide.

In another embodiment, the present invention provides novel epothilonederivative compounds in substantially pure form useful in agriculture,veterinary practice, and medicine. In one embodiment, the compounds areuseful as fungicides. In another embodiment, the compounds are useful incancer chemotherapy. In a preferred embodiment, the compound is anepothilone derivative that is at least as potent against tumor cells asepothilone B or D. In another embodiment, the compounds are useful asimmunosuppressants. In another embodiment, the compounds are useful inthe manufacture of another compound. In a preferred embodiment, thecompounds are formulated in a mixture or solution for administration toa human or animal.

In another embodiment, the present invention provides a method oftreating cancer, which method comprises administering a therapeuticallyeffective amount of a novel epothilone compound of the invention.

These and other embodiments of the invention are described in moredetail in the following description, the examples, and claims set forthbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a number of precursor compounds to N-acetyl cysteaminethioester derivatives that can be supplied to an epothilone PKS of theinvention in which the NRPS-like module one or module 2 KS domain hasbeen inactivated to produce a novel epothilone derivative. A generalsynthetic procedure for making such compounds is also shown.

FIG. 2 shows restriction site and function maps of plasmids pKOS35-82.1and pKOS35-82.2.

FIG. 3 shows restriction site and function maps of plasmids pKOS35454and pKOS90-22.

FIG. 4 shows a schematic of a protocol for introducing the epothilonePKS and modification enzyme genes into the chromosome of a Myxococcusxanthus host cell as described in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides recombinant host cellsof the suborder Cystobacterineae containing recombinant expressionvectors that encode heterologous PKS genes and produce polyketidessynthesized by the PKS enzymes encoded on those vectors. As used herein,the term recombinant refers to a compound or composition produced byhuman intervention, typically by specific and directed manipulation of agene or portion thereof. The suborder Cystobacterineae is one of two(the other is Sorangineae, which includes the epothilone producerSorangium cellulosum) in the order Myxococcales. The suborderCystobacterineae includes the family Myxococcaceae and the familyCystobacteraceae. The family Myxococcacceae includes the genusAngiococcus (i.e., A. disciformis), the genus Myxococcus, and the genusCorallococcus (i.e., C. macrosporus, C. corralloides, and C. exiguus).The family Cystobacteraceae includes the genus Cystobacter (i.e., C.fuscus, C. ferrugineus, C. minor, C. velatus, and C. violaceus), thegenus Melittangium (i.e., M. boletus and M. lichenicola), the genusStigmatella (i.e., S. erecta and S. aurantiaca), and the genusArchangium (i.e., A. gephyra). Especially preferred host cells of theinvention are those that produce a polyketide at equal to or greaterthan 10 to 20 mg/L, more preferably at equal to or greater than 100 to200 mg/L, and most preferably at equal to or greater than 1 to 2 g/L.

In a preferred embodiment, the host cells of the invention are from thegenus Myxococcus or the genus Stigmatella. In especially preferredembodiments, the host cells are selected from the group consisting of M.stipitatus, M. fulvus, M. xanthus, M. virescens, S. erecta, and S.aurantiaca. Especially preferred Myxococcus host cells of the inventionare those that produce a polyketide at equal to or greater than 10 to 20mg/L, more preferably at equal to or greater than 100 to 200 mg/L, andmost preferably at equal to or greater than 1 to 2 g/L. Especiallypreferred are M. xanthus host cells that produce at these levels. M.xanthus host cells that can be employed for purposes of the inventioninclude the DZ1 cell line (Campos et al., 1978, J. Mol. Biol.119:167-178, incorporated herein by reference), the TA-producing cellline ATCC 31046, the DK1219 cell line (Hodgkin and Kaiser, 1979, Mol.Gen. Genet. 171:177-191, incorporated herein by reference), and theDK1622 cell line (Kaiser, 1979, Proc. Natl. Acad. Sci. USA 76:5952-5956, incorporated herein by reference).

The host cells of the invention comprise a recombinant DNA expressionvector, and in another embodiment, the present invention providesrecombinant DNA vectors capable of chromosomal integration orextrachromosomal replication in these host cells. The vectors of theinvention comprise at least a portion of a PKS coding sequence and arecapable of directing expression of a functional PKS enzyme in the hostcells of the invention. As used herein, the term expression vectorrefers to any nucleic acid that can be introduced into a host cell. Anexpression vector can be maintained stably or transiently in a cell,whether as part of the chromosomal or other DNA in the cell or in anycellular compartment, such as a replicating vector in the cytoplasm. Anexpression vector also comprises a gene that serves to direct thesynthesis RNA that is translated into a polypeptide in the cell or cellextract. Thus, the vector either includes a promoter to enhance geneexpression or is integrated into a site in the chromosome such that geneexpression is obtained. Furthermore, expression vectors typicallycontain additional functional elements, such as resistance-conferringgenes to act as selectable markers and regulatory genes to enhancepromoter activity.

Typically, the expression vector will comprise one or more marker genesby which host cells containing the vector can be identified and/orselected. Illustrative antibiotic resistance conferring genes for use invectors of the invention include the ermE (confers resistance toerythromycin and lincomycin), tsr (confers resistance to thiostrepton),aadA (confers resistance to spectinomycin and streptomycin), aacC4(confers resistance to apramycin, kanamycin, gentamicin, geneticin(G418), and neomycin), hyg (confers resistance to hygromycin), and vph(confers resistance to viomycin) resistance conferring genes. Selectablemarkers for use in Myxococcus xanthus include kanamycin, tetracycline,chloramphenicol, zeocin, spectinomycin, and streptomycin resistanceconferring genes.

The various components of an expression vector can vary widely,depending on the intended use of the vector. In particular, thecomponents depend on the host cell(s) in which the vector will be usedand the manner in which it is intended to function. For example, certainpreferred vectors of the invention are integrating vectors: the vectorsintegrate into the chromosomal DNA of the host cell. Such vectors cancomprise a phage attachment site or DNA segments complementary tosegments of the host cell chromosomal DNA to direct integration.Moreover, and as exemplified herein, a series of such vectors can beused to build the PKS gene cluster in the host cell, with each vectorcomprising only a portion of the complete PKS gene cluster. Thus, therecombinant DNA expression vectors of the invention may comprise only aportion of a PKS gene. Homologous recombination can also be used todelete, disrupt, or alter a gene, including a heterologous PKS genepreviously introduced into the host cell.

In a preferred embodiment, the present invention provides expressionvectors and recombinant Myxococcus, preferably M. xanthus, host cellscontaining those expression vectors that produce a polyketide.Presently, vectors that replicate extrachromosomally in M. xanthus arenot known. There are, however, a number of phage known to integrate intoM. xanthus chromosomal DNA, including Mx8, Mx9, Mx81, and Mx82. Theintegration and attachment functions of these phages can be placed onplasmids to create phage-based expression vectors that integrate intothe M. xanthus chromosomal DNA. Of these, phage Mx9 and Mx8 arepreferred for purposes of the present invention. Plasmid pPLH343,described in Salmi et al., February 1998, Genetic determinants ofimmunity and integration of temperate Myxococcus xanthus phage Mx8, J.Bact. 180(3): 614-621, is a plasmid that replicates in E. coli andcomprises the phage Mx8 genes that encode the attachment and integrationfunctions.

A wide variety of promoters are available for use in the preferredMyxococcus expression vectors of the invention. See Example 8, below.For example, the promoter of the epothilone PKS gene (see U.S. patentapplication Ser. No. 09/443,501, filed 19 Nov. 1999, incorporated hereinby reference) functions in M. xanthus host cells. The promoter can beused to drive expression of one or more epothilone PKS genes or anotherPKS gene product in recombinant host cells. Another preferred promoterfor use in Myxococcus xanthus host cells for purposes of expressing arecombinant PKS of the invention is the promoter of the pilA gene of M.xanthus. This promoter, as well as two M. xanthus strains that expresshigh levels of gene products from genes controlled by the pilA promoter,a pilA deletion strain and a pilS deletion strain, are described in Wuand Kaiser, December 1997, Regulation of expression of the pilA gene inMyxococcus xanthus, J. Bact. 179(24):7748-7758, incorporated herein byreference. The invention present invention also provides recombinantMyxococcus host cells comprising both the pilA and pilS deletions.Another preferred promoter is the starvation dependent promoter of thesdcK gene.

The present invention provides preferred expression vectors for use inpreparing the recombinant Myxococcus xanthus expression vectors and hostcells of the invention. These vectors, designated plasmids pKOS35-82.1and pKOS35-82.2 (FIG. 2), are able to replicate in E. coli host cells aswell as integrate into the chromosomal DNA of M. xanthus. The vectorscomprise the Mx8 attachment and integration genes as well as the pilApromoter with restriction enzyme recognition sites placed convenientlydownstream. The two vectors differ from one another merely in theorientation of the pilA promoter on the vector and can be readilymodified to include the epothilone PKS and modification enzyme genes ofthe invention. The construction of the vectors is described in Example1.

In another embodiment, the present invention provides a method forproducing a polyketide in a host cell of the suborder Cystobacterineae,which polyketide is not naturally produced in said host cell, saidmethod comprising culturing the host cell transformed with a recombinantDNA vector of the invention under conditions such that a PKS geneencoded on the vector is expressed and said polyketide is produced. Withthis method, any of the diverse members of the polyketides produced bymodular PKS enzymes can be prepared. In addition, novel polyketidesderived from hybrid or other recombinant PKS genes can also be preparedusing this method. In a preferred embodiment, the PKS genes encode amodular PKS.

A large number of modular PKS genes have been cloned and are immediatelyavailable for use in the vectors and methods of the invention. Thepolyketides produced by PKS enzymes are often further modified bypolyketide modification enzymes, called tailoring enzymes, thathydroxylate, epoxidate, methylate, and glycosylate the polyketideproduct of the PKS. In accordance with the methods of the invention,these genes can also be introduced into the host cell to prepare amodified polyketide of interest. The following Table lists referencesdescribing illustrative PKS genes and corresponding enzymes that can beutilized in the construction of the recombinant PKSs and thecorresponding DNA compounds that encode them of the invention. Alsopresented are various references describing polyketide tailoring andmodification enzymes and corresponding genes that can be employed tomake the recombinant DNA compounds of the present invention.

PKS and Polyketide Tailoring Enzyme Genes Avermectin

U.S. Pat. No. 5,252,474; U.S. Pat. No. 4,703,009; and EP Pub. No.118,367 to Merck.

MacNeil et al., 1993, Industrial Microorganisms: Basic and AppliedMolecular Genetics, Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256,A Comparison of the Genes Encoding the Polyketide Synthases forAvermectin, Erythromycin, and Nemadectin.

MacNeil et al., 1992, Gene 115: 119-125, Complex Organization of theStreptomyces avermtilis genes encoding the avermectin polyketidesynthase.

Ikeda and Omura, 1997, Chem. Res. 97: 2599-2609, Avermectinbiosynthesis.

Candicidin (FR008)

Hu et al., 1994. Mol. Microbiol. 14: 163-172.

Epothilone

PCT Pub. No. 99/66028 to Novartis.

PCT Pat. App. No. US99/27438 to Kosan.

Erythromycin

PCT Pub. No. 93/13663; U.S. Pat. No. 6,004,787; and U.S. Pat. No.5,824,513 to Abbott.

Donadio et al., 1991, Science 252:675-9.

Cortes et al., 8 Nov. 1990, Nature 348:1768, An unusually largemultifunctional polypeptide in the erythromycin producing polyketidesynthase of Saccharopolyspora erythraea.

Glycosylation Enzymes

PCT Pub. No. 97/23630 and U.S. Pat. No. 5,998,194 to Abbott.

FK-506

Motamedi et al., 1998, The biosynthetic gene cluster for themacrolactone ring of the immunosuppressant FK-506, Eur. J. biochem. 256:528-534.

Motamedi et al., 1997, Structural organization of a multifunctionalpolyketide synthase involved in the biosynthesis of the macrolideimmunosuppressant FK-506, Eur. J. Biochem. 244: 74-80.

Methyltransferase

U.S. Pat. No. 5,264,355 and U.S. Pat. No. 5,622,866 to Merck.

Motamedi et al., 1996, Characterization of methyltransferase andhydroxylase genes involved in the biosynthesis of the immunosuppressantsFK-506 and FK-520, J. Bacteriol. 178: 5243-5248.

FK-520

PCT Pub. No. 00/20601 and U.S. patent application Ser. No. 09/410,551,filed 1 Oct. 1999 to Kosan.

Nielsen et al., 1991, Biochem. 30:5789-96.

Lovastatin

U.S. Pat. No. 5,744,350 to Merck.

Narbomycin

U.S. patent application Ser. No. 09/434,288, filed 5 Nov. 1999 to Kosan.

Nemadectin

MacNeil et al., 1993, supra.

Niddamycin

PCT Pub. No. 98/51695 to Abbott.

Kakavas et al., 1997, Identification and characterization of theniddamycin polyketide synthase genes from Streptomyces caelestis, J.Bacteriol. 179: 7515-7522.

Oleandomycin

Swan et al., 1994, Characterisation of a Streptomyces antibioticus geneencoding a type I polyketide synthase which has an unusual codingsequence, Mol. Gen. Genet. 242: 358-362.

U.S. patent application Ser. No. 09/428,517, filed 28 Oct. 1999 toKosan.

Olano et al., 1998, Analysis of a Streptomyces antibioticus chromosomalregion involved in oleandomycin biosynthesis, which encodes twoglycosyltransferases responsible for glycosylation of the macrolactonering, Mol. Gen. Genet. 259(3): 299-308.

PCT Pat. App. Pub. No. WO 99/05283 to Hoechst.

Picromycin

PCT Pub. No. 99/61599 to Kosan.

PCT Pub. No. 00/00620 to the University of Minnesota.

Xue et al., 1998, Hydroxylation of macrolactones YC-17 and narbomycin ismediated by the pikC-encoded cytochrome P450 in Streptomyces venezuelae,Chemistry & Biology 5(11): 661-667.

Xue et al., Oct. 1998, A gene cluster for macrolide antibioticbiosynthesis in Streptomyces venezuelae: Architecture of metabolicdiversity, Proc. Natl. Acad. Sci. USA 95:12111 12116.

Platenolide

EP Pub. No. 791,656; and U.S. Pat. No. 5,945,320 to Lilly.

Rapamycin

Schwecke et al., August 1995, The biosynthetic gene cluster for thepolyketide rapamycin, Proc. Natl. Acad. Sci. USA 92:7839-7843.

Aparicio et al., 1996, Organization of the biosynthetic gene cluster forrapamycin in Streptomyces hygroscopicus: analysis of the enzymaticdomains in the modular polyketide synthase, Gene 169:9-16.

Rifamycin

PCT Pub. No. WO 98/07868 to Novartis.

August et al., 13 Feb. 1998, Biosynthesis of the ansamycin antibioticrifamycin: deductions from the molecular analysis of the rifbiosynthetic gene cluster of Amycolatopsis mediterranei S669, Chemistry& Biology, 5(2): 69-79.

Sorangium PKS

U.S. patent application Ser. No. 09/144,085, filed 31 Aug. 1998 toKosan.

Soraphen

U.S. Pat. No. 5,716,849 to Novartis.

Schupp et al., 1995, J. Bacteriology 177: 3673-3679. A Sorangiumcellulosum (Myxobacterium) Gene Cluster for the Biosynthesis of theMacrolide Antibiotic Soraphen A: Cloning, Characterization, and Homologyto Polyketide Synthase Genes from Actinomycetes.

Spinocyn

PCT Pub. No. 99/46387 to DowElanco.

Spiramycin

U.S. Pat. No. 5,098,837 to Lilly.

Activator Gene

U.S. Pat. No. 5,514,544 to Lilly.

Tylosin

U.S. Pat. No. 5,876,991; U.S. Pat. No. 5,672,497; U.S. Pat. No.5,149,638; EP Pub. No. 791,655; and EP Pub. No. 238,323 to Lilly.

Kuhstoss et al., 1996, Gene 183:231-6., Production of a novel polyketidethrough the construction of a hybrid polyketide synthase.

Tailoring Enzymes

Merson-Davies and Cundliffe, 1994, Mol. Microbiol. 13: 349-355. Analysisof five tylosin biosynthetic genes from the tylBA region of theStreptomyces fradiae genome.

Any of the above genes, with or without the genes for polyketidemodification, if any, can be employed in the recombinant DNA expressionvectors of the invention. Moreover, the host cells of the invention canbe constructed by transformation with multiple vectors, each containinga portion of the desired PKS and modification enzyme gene cluster; seeU.S. Pat. No. 6,033,883, incorporated herein by reference.

For improved production of a polyketide in a host cell of the invention,including Myxococcus host cells, one can also transform the cell toexpress a heterologous phosphopantetheinyl transferase. PKS proteinsrequire phosphopantetheinylation of the ACP domains of the loading andextender modules as well as of the PCP domain of any NRPS.Phosphopantetheinylation is mediated by enzymes calledphosphopantetheinyl transferases (PPTases). To produce functional PKSenzyme in host cells that do not naturally express a PPTase able to acton the desired PKS enzyme or to increase amounts of functional PKSenzyme in host cells in which the PPTase is limiting, one can introducea heterologous PPTase, including but not limited to Sfp, as described inPCT Pub. Nos. 97/13845 and 98/27203, and U.S. patent application Ser.No. 08/728,742, filed 11 Oct. 1996, and U.S. Pat. No. 6,033,883, each ofwhich is incorporated herein by reference.

The host cells of the invention can be used not only to produce apolyketide found in nature but also to produce polyketides produced fromrecombinant PKS genes and modification enzymes. In one importantembodiment, the present invention provides recombinant DNA expressionvectors that comprise a hybrid PKS. For purposes of the presentinvention a hybrid PKS is a recombinant PKS that comprises all or partof one or more extender modules, loading module, andthioesterase/cyclase domain of a first PKS and all or part of one ormore extender modules, loading module, and thioesterase/cyclase domainof a second PKS.

Those of skill in the art will recognize that all or part of either thefirst or second PKS in a hybrid PKS of the invention need not beisolated from a naturally occurring source. For example, only a smallportion of an AT domain determines its specificity. See U.S. patentapplication Ser. No. 09/346,860 and PCT Pub. No. 00/01838, each of whichis incorporated herein by reference. The state of the art in DNAsynthesis allows the artisan to construct de novo DNA compounds of sizesufficient to construct a useful portion of a PKS module or domain. Forpurposes of the present invention, such synthetic DNA compounds aredeemed to be a portion of a PKS.

As the above Table illustrates, there are a wide variety of PKS genesthat serve as readily available sources of DNA and sequence informationfor use in constructing the hybrid PKS-encoding DNA compounds of theinvention. Methods for constructing hybrid PKS-encoding DNA compoundsare described in U.S. Pat. Nos. 6,022,731; 5,672,491; and 5,712,146 andU.S. patent application Ser. No. 09/073,538, filed 6 May 1998, and09/141,908, filed 28 Aug. 1998, each of which is incorporated herein byreference. The hybrid PKS-encoding DNA compounds of the invention can beand often are hybrids of more than two PKS genes. Even where only twogenes are used, there are often two or more modules in the hybrid genein which all or part of the module is derived from a second (or third)PKS gene. Those of skill in the art will appreciate that a hybrid PKS ofthe invention includes but is not limited to a PKS of any of thefollowing types: (i) a PKS that contains a module in which at least oneof the domains is from a heterologous module; (ii) a PKS that contains amodule from a heterologous PKS; (iii) a PKS that contains a protein froma heterologous PKS; and (iv) combinations of the foregoing.

Hybrid PKS enzymes of the invention are often constructed by replacingcoding sequences for one or more domains of a module from a first PKSwith coding sequences for one or more domains of a module from a secondPKS to construct a recombinant coding sequence. Generally, any referenceherein to inserting or replacing a KR, DH, and/or ER domain includes thereplacement of the associated KR, DH, or ER domains in that module,typically with corresponding domains from the module from which theinserted or replacing domain is obtained. The KS and/or ACP of anymodule can also be replaced, if desired or beneficial, with another KSand/or ACP. In each of these replacements or insertions, theheterologous KS, AT, DH, KR, ER, or ACP coding sequence can originatefrom a coding sequence from another module of the same or different PKSor from chemical synthesis to obtain the hybrid PKS coding sequence.

While an important embodiment of the present invention relates to hybridPKS genes, the present invention also provides recombinant PKS genes inwhich there is no second PKS gene sequence present but which differ froma naturally occurring PKS gene by one or more deletions. The deletionscan encompass one or more modules or domains and/or can be limited to adeletion within one or more modules or domains. When a deletionencompasses an entire extender module (other than an NRPS module), theresulting polyketide derivative is at least two carbons shorter than thecompound produced from the PKS from which the deleted version wasderived. The deletion can also encompass an NRPS module and/or a loadingmodule. When a deletion is within a module, the deletion typicallyencompasses a KR, DH, or ER domain, or both DH and ER domains, or bothKR and DH domains, or all three KR, DH, and ER domains.

To construct any PKS of the invention, one can employ a technique,described in PCT Pub. No. 98/27203 and U.S. Pat. No. 6,033,883, each ofwhich is incorporated herein by reference, in which the various genes ofthe PKS and optionally genes for one or more polyketide modificationenzymes are divided into two or more, often three, segments, and eachsegment is placed on a separate expression vector (see also, U.S. patentapplication Ser. No. 09/______, filed 14 Apr. 2000 (attorney docket no.30062-20041, which claims priority to Ser. No. 60/129,731, filed 16 Apr.1999, both of which are incorporated herein by reference). In thismanner, the full complement of genes can be assembled and manipulatedmore readily for heterologous expression, and each of the segments ofthe gene can be altered, and various altered segments can be combined ina single host cell to provide a recombinant PKS gene of the invention.This technique makes more efficient the construction of large librariesof recombinant PKS genes, vectors for expressing those genes, and hostcells comprising those vectors. In this and other contexts, the genesencoding the desired PKS not only can be present on two or more vectors,but also can be ordered or arranged differently from that which existsin the native producer organism from which the genes were derived.

In a preferred and illustrative embodiment, the recombinant host cell ofthe invention produces epothilone or an epothilone derivative. Theepothilones (epothilone A, B, C, D, E, and F) and compounds structurallyrelated thereto (epothilone derivatives) are potent cytotoxic agentsspecific for eukaryotic cells. These compounds have application asanti-fungals, cancer chemotherapeutics, and immunosuppressants. Theepothilones are produced at very low levels in the naturally occurringSorangium cellulosum cells in which they have been identified. Moreover,S. cellulosum is very slow growing, and fermentation of S. cellulosumstrains is difficult and time-consuming. One important benefit conferredby the present invention is the ability simply to produce an epothiloneor epothilone derivative in a non-S; cellulosum host cell. Anotheradvantage of the present invention is the ability to produce theepothilones at higher levels and in greater amounts in the recombinanthost cells provided by the invention than possible in the naturallyoccurring epothilone producer cells. Yet another advantage is theability to produce an epothilone derivative in a recombinant host cell.Thus, the present invention provides recombinant host cells that producea desired epothilone or epothilone derivative. In a preferredembodiment, the host cell produces the epothilones at equal to orgreater than 10 mg/L. In one embodiment, the invention provides hostcells that produce one or more of the epothilones or epothilonederivatives at higher levels than produced in the naturally occurringorganisms that produce epothilones. In another embodiment, the inventionprovides host cells that produce mixtures of epothilones that are lesscomplex than the mixtures produced by naturally occurring host cellsthat produce epothilones.

In an especially preferred embodiment, the host cells of the inventionproduce less complex mixtures of epothilones than do naturally occurringcells that produce epothilones. Naturally occurring cells that produceepothilones typically produce a mixture of epothilones A, B, C, D, E,and F. The table below summarizes the epothilones produced in differentillustrative host cells of the invention.

Cell Type Epothilones Produced Epothilones Not Produced 1 A, B, C, D E,F 2 A, C B, D, E, F 3 B, D A, C, E, F 4 B A, C, D, E, F 5 D A, B, C, E,FThus, the recombinant host cells of the invention also include hostcells that produce only one desired epothilone or epothilone derivative.

An analysis of the domains of the epothilone PKS suggests that the PKSenzyme catalyzes the production of epothilones G and H, which differfrom one another in that epothilone G has a hydrogen at C-12 andepothilone H has a methyl group at that position. The variance at theC-12 position is predicted to arise from the ability of thecorresponding AT domain (extender module 4) of the PKS to bind eithermalonyl CoA, leading to hydrogen, or methylmalonyl CoA, leading tomethyl. However, epothilones G and H have not been observed in nature orin the recombinant host cells of the invention; instead, the products ofthe PKS appear to be epothilones C and D, which differ from epothilonesG and H, respectively, by having a C-12 to C-13 double bond and lackinga C-13 hydroxyl substituent. Thus, the dehydration reaction that wouldform epothilones C and D from epothilones G and H may be carried out bythe epothilone PKS itself or by another enzymatic activity that ispresent in the host cells in which the epothilones have been produced todate. Epothilones A and B are formed from epothilones C and D,respectively, by epoxidation of the C-12 to C-13 double bond by the epoKgene product. Epothilones E and F are formed from epothilones A and B,respectively, by hydroxylation of the C-21 methyl group.

Thus expression of the epothilone PKS genes and the epoK gene in a hostcell of the invention leads to the production of epothilones A, B, C,and D. If the epoK gene is not present or is rendered inactive bymutation, then only epothilones C and D are produced. If the AT domainof extender module 4 is replaced by an AT domain specific for malonyl CoA, then epothilones A and C only are produced, and if there is nofunctional epoK gene, then only epothilone C is produced. If the ATdomain of extender module 4 is replaced by an AT domain specific formethylmalonyl Co A, then epothilones B and D only are produced, and ifthere is no functional epoK gene, then only epothilone D is produced.

The epothilone PKS and modification enzyme genes were cloned from theepothilone producing strain, Sorangium cellulosum SMP44. Total DNA wasprepared from this strain using the procedure described by Jaoua et al.,1992, Plasmid 28:157-165, incorporated herein by reference. A cosmidlibrary was prepared from S. cellulosum genomic DNA in pSupercos(Stratagene). The entire PKS and modification enzyme gene cluster wasisolated in four overlapping cosmid clones (deposited with the AmericanType Culture Collection (ATCC), Manassas, Va., USA, and assigned ATCCaccession numbers as follows pKOS35-70.1A2 (ATCC 203782), pKOS35-70.4(ATCC 203781), pKOS35-70.8A3 (ATCC 203783), and pKOS35-79.85 (ATCC203780)) and the DNA sequence determined, as set forth in U.S. patentapplication Ser. No. 09/443,501, filed 19 Nov. 1999, incorporated hereinby reference. DNA sequence analysis revealed a PKS gene cluster with aloading module and nine extender modules. Downstream of the PKS sequenceis an open reading frame (ORF), designated epoK, that shows stronghomology to cytochrome P450 oxidase-genes and encodes the epothiloneepoxidase modification enzyme.

The PKS genes are organized in 6 ORFs. At the polypeptide level, theloading module and extender modules 1 (an NRPS), 2, and 9 appear onindividual polypeptides; their corresponding genes are designated epoA,epoB, epoC and epoF respectively. Modules 3, 4, 5, and 6 are containedon a single polypeptide whose gene is designated epoD, and modules 7 and8 are on another polypeptide whose gene is designated epoE. It is clearfrom the spacing between ORFs that epoC, epoD, epoE and epoF constitutean operon. The epoA, epoB, and epoK gene may be also part of the largeoperon, but there are spaces of approximately 100 bp between epoB andepoC and 115 bp between epoF and epoK which could contain a promoter.The epothilone PKS gene cluster is shown schematically below.

Immediately downstream of epoK, the P450 epoxidase gene, is ORF1, whichencodes a polypeptide that appears to include membrane spanning domainsand may be involved in epothilone transport. This ORF is followed by anumber of ORFs that include genes that may encode proteins involved intransport and regulation.

A detailed examination of the modules shows an organization andcomposition consistent with one able to be used for the biosynthesis ofepothilone. The description that follows is at the polypeptide level.The sequence of the AT domain in the loading module and in extendermodules 3, 4, 5, and 9 shows similarity to the consensus sequence formalonyl loading modules, consistent with the presence of an H side chainat C-14, C-12 (epothilones A and C), C-10, and C-2, respectively, aswell as the loading module. The AT domains in modules 2, 6, 7, and 8resemble the consensus sequence for methylmalonyl specifying AT domains,again consistent with the presence of methyl side chains at C-16, C-8,C-6, and C-4 respectively.

The loading module contains a KS domain in which the cysteine residueusually present at the active site is instead a tyrosine. This domain isdesignated as KS^(y) and serves as a decarboxylase, which is part of itsnormal function, but cannot function as a condensing enzyme. Thus, theloading module is expected to load malonyl CoA, move it to the ACP, anddecarboxylate it to yield the acetyl residue required for condensationwith cysteine. Module 1 is the non-ribosomal peptide synthetase thatactivates cysteine and catalyzes the condensation with acetate on theloading module. The sequence contains segments highly similar toATP-binding and ATPase domains, required for activation of amino acids,a phosphopantotheinylation site, and an elongation domain. Module 2determines the structure of epothilone at C-15-C-17. The presence of theDH domain in module 2 yields the C-16-17 dehydro moiety in the molecule.The domains in module 3 are consistent with the structure of epothiloneat C-14 and C-15; the OH that comes from the action of the KR isemployed in the lactonization of the molecule. Module 4 controls thestructure at C-12 and C-13 where a double bond is found in epothilones Cand D. Although the sequence of the AT domain appears to resemble thosethat specify malonate loading, it can also load methylmalonate, therebyaccounting in part for the mixture of epothilones found in thefermentation broths of the naturally producing organisms.

A significant departure from the expected array of functions was foundin module 4. This module was expected to contain a DH domain, therebydirecting the synthesis of epothilones C and D as the products of thePKS. Rigorous analysis revealed that the space between the AT and KRdomains of module 4 was not large enough to accommodate a functional DHdomain. Thus, the extent of reduction at module 4 does not proceedbeyond the ketoreduction of the beta-keto formed after the condensationdirected by module 4. Because the C-12,13 unsaturation has beendemonstrated (epothilones C and D), there must be an additionaldehydratase function that introduces the double bond. The dehydrationreaction that mediates the formation of this double bond may be due tothe action of an as yet unrecognized domain of the epothilone PKS (forexample, dehydration could occur in the next module, which possesses anactive DH domain and could generate a conjugated diene precursor priorto its dehydrogenation by an ER domain) or an endogenous enzyme in thehost cells in which it is observed. As shown herein, the PKS genes andflanking sequences are sufficient to confer the ability to produceepothilones C and D in a host cell of the invention.

Thus, the action of the dehydratase could occur either during thesynthesis of the polyketide or after cyclization has taken place. In theformer case, the compounds produced at the end of acyl chain growthwould be epothilones C and D. If the C-12,13 dehydration were apost-polyketide event, the completed acyl chain would have a hydroxylgroup at C-13, as shown below. The names epothilones G and H have beenassigned to the 13-hydroxy compounds produced in the absence of or priorto the action of the dehydratase.

Modules 5 and 6 each have the full set of reduction domains (KR, DH andER) to yield the methylene functions at C-11 and C-9. Modules 7 and 9have KR domains to yield the hydroxyls at C-7 and C-3, and module 8 doesnot have a functional KR domain, consistent with the presence of theketo group at C-5. Module 8 also contains a methyltransferase (MT)domain that results in the presence of the geminal dimethyl function atC-4. Module 9 has a thioesterase domain that terminates polyketidesynthesis and catalyzes ring closure.

The genes, proteins, modules, and domains of the epothilone PKS aresummarized in the following Table.

Gene Protein Modules Domains Present epoA EpoA Load KS^(Y) mAT ER ACPepoB EpoB 1 NRPS, condensation, heterocyclization, adenylation,thiolation, PCP epoC EpoC 2 KS mmAT DH KR ACP epoD EpoD 3-6 KS mAT KRACP; KS mAT KR ACP; KS mAT DH ER KR ACP; KS mmAT DH ER KR ACP epoE EpoE7-8 KS mmAT KR ACP; KS mmAT MT DH* KR* ACP epoF EpoF 9 KS mAT KR DH* ER*ACP TE NRPS—non-ribosomal peptide synthetase; KS—ketosynthase;mAT—malonyl CoA specifying acyltransferase; mmAT—methylmalonyl CoAspecifying acyltransferase; DH—dehydratase; ER—enoylreductase;KR—ketoreductase; MT—methyltransferase; TE thioesterase; *inactivedomain.Inspection of the sequence has revealed translational coupling betweenepoA and epoB (loading module and module 1) and between epoC and epoD.Very small gaps are seen between epoD and epoE and epoE and epoF butgaps exceeding 100 bp are found between epoB and epoC and epoF and epoK.These intergenic regions may contain promoters.

Thus, the epothilone PKS is multiprotein complex composed of the geneproducts of the epoA, epoB, epoC, epoD, epoE, and epoF genes. To conferthe ability to produce epothilones to a host cell, one provides the hostcell with the recombinant epoA, epoB, epoC, epoD, epoE, and epoF genesof the present invention, and optionally other genes, capable ofexpression in that host cell. Those of skill in the art will appreciatethat, while the epothilone and other PKS enzymes may be referred to as asingle entity herein, these enzymes are typically multisubunit proteins.Thus, one can make a derivative PKS (a PKS that differs from a naturallyoccurring PKS by deletion or mutation) or hybrid PKS (a PKS that iscomposed of portions of two different PKS enzymes) by altering one ormore genes that encode one or more of the multiple proteins thatconstitute the PKS.

The post-PKS modification or tailoring of epothilone includes multiplesteps mediated by multiple enzymes. These enzymes are referred to hereinas tailoring or modification enzymes. Expression of the epothilone PKSgenes epoA, epoB, epoC, epoD, epoE, and epoF in certain host cells ofthe invention that do not express epoK leads to the production ofepothilones C and D, which lack the C-12-C-13 epoxide of epothilones Aand B, having instead a C-12-C-13 double bond. Thus, epothilones C and Dare converted to epothilones A and B by an epoxidase encoded by the epoKgene. Epothilones A and B are converted to epothilones E and F by ahydroxylase gene, which may be encoded by a gene associated with theepothilone PKS gene cluster or by another gene endogenous to Sorangiumcellulosum. Thus, one can produce an epothilone or epothilone derivativemodified as desired in a host cell by providing that host cell with oneor more recombinant modification enzyme genes provided by the inventionor by utilizing a host cell that naturally expresses (or does notexpress) the modification enzyme.

Thus, the present invention provides a wide variety of recombinant DNAcompounds and host cells for expressing the naturally occurringepothilones A, B, C, and D and derivatives thereof. The invention alsoprovides recombinant host cells that produce epothilone derivativesmodified in a manner similar to epothilones E and F. Moreover, theinvention provides host cells that can produce epothilones G and H,either by expression of the epothilone PKS genes in host cells that donot express the dehydratase that converts epothilones G and H to C and Dor by mutating or altering the PKS to abolish the dehydratase function,if it is present in the epothilone PKS.

The present invention also provides a wide variety of recombinant DNAcompounds and host cells that make epothilone derivatives. As usedherein, the phrase epothilone derivative refers to a compound that isproduced by a recombinant epothilone PKS in which at least one domainhas been inserted or either rendered inactive, mutated to alter itscatalytic function, or replaced by a domain with a different function.In any event, the epothilone derivative PKS so produced functions toproduce a compound that differs in structure from a naturally occurringepothilone and so is called an epothilone derivative. To facilitate abetter understanding of the recombinant DNA compounds and host cellsprovided by the invention, a detailed discussion of the loading moduleand each of the modules of the epothilone PKS, as well as novelrecombinant derivatives thereof, is provided below.

The loading module of the epothilone PKS includes an “inactive” KSdomain, designated KS^(Y), that, due to the presence of a tyrosine (Y)residue in place of the cysteine residue found in “active” KS domains,is unable to perform the condensation reaction mediated by active KSdomains. The KS^(Y) domain does carry out the decarboxylation reactionmediated by KS domains. Such “inactive” KS domains are found in otherPKS enzymes, usually with a glutamine (Q) residue in place of the activesite cysteine, and are called KS^(Q) domains. The KS^(Q) domain in ratfatty acid synthase has been shown to be unable to perform condensationbut exhibits a 2 order of magnitude increase in decarboxylation. SeeWitkowski et al., 7 Sep. 1999, Biochem. 38(36): 11643-11650,incorporated herein by reference. A KS^(Q) domain may be more efficientat decarboxylation than a KS^(Y) domain, so the replacement of theKS^(Y) domain in the epothilone PKS with a KS^(Q) domain may increasethe efficiency of epothilone biosynthesis. This can be accomplishedmerely by changing the codon from a tyrosine to a glutamine codon, asdescribed in Example 6, below. This can also be accomplished byreplacing the KS^(Y) domain with a KS^(Q) domain of another PKS, such asthe oleandolide PKS or the narbonolide PKS (see the references cited inthe Table above in connection with the oleandomycin, narbomycin, andpicromycin PKS and modification enzymes).

The epothilone loading module also contains an AT domain specific formalonyl CoA (which is believed to be decarboxylated by the KS^(Y) domainto yield an acetyl group), and an ACP domain. The present inventionprovides recombinant epothilone derivative loading modules or theirencoding DNA sequences in which the malonyl specific AT domain or itsencoding sequence has been changed to another specificity, such asmethylmalonyl CoA, ethylmalonyl CoA, and 2-hydroxymalonyl CoA. Whenexpressed with the other proteins of the epothilone PKS, such loadingmodules lead to the production of epothilones in which the methylsubstituent of the thiazole ring of epothilone is replaced with,respectively, ethyl, propyl, and hydroxymethyl. The present inventionprovides recombinant PKS enzymes comprising such loading modules andhost cells for producing such enzymes and the polyketides producedthereby. An AT domain specific for 2-hydroxymalonyl CoA will result in apolyketide with a hydroxyl group at the corresponding location in thepolyketide produced; the hydroxyl group can be methylated to yield amethoxy group by polyketide modification enzymes. See, e.g., thereferences cited in connection with the FK-520 PKS in the Table above.Consequently, reference to a PKS that has a 2-hydroxymalonyl specific ATdomain herein similarly refers to polyketides produced by that PKS thathave either a hydroxyl or methoxyl group at the corresponding locationin the polyketide.

The loading module of the epothilone PKS also comprises an ER domain.While, this ER domain may be involved in forming one of the double bondsin the thiazole moiety in epothilone (in the reverse of its normalreaction), it may be non-functional. In either event, the inventionprovides recombinant DNA compounds that encode the epothilone PKSloading module with and without the ER region, as well as hybrid loadingmodules that contain an ER domain from another PKS (either active orinactive, with or without accompanying KR and DH domains) in place ofthe ER domain of the epothilone loading module. The present inventionalso provides recombinant PKS enzymes comprising such loading modulesand host cells for producing such enzymes and the polyketides producedthereby.

The loading module of the epothilone PKS is followed by the firstextender module of the PKS, which is an NRPS module specific forcysteine. This NRPS module is naturally expressed as a discrete protein,the product of the epoB gene. In one embodiment, a portion of the NRPSmodule coding sequence is utilized in conjunction with a heterologouscoding sequence. In this embodiment, the invention provides, forexample, changing the specificity of the NRPS module of the epothilonePKS from a cysteine to another amino acid. This change is accomplishedby constructing a coding sequence in which all or a portion of theepothilone PKS NRPS module coding sequences have been replaced by thosecoding for an NRPS module of a different specificity. In oneillustrative embodiment, the specificity of the epothilone NRPS moduleis changed from cysteine to serine or threonine. When the thus modifiedNRPS module is expressed with the other proteins of the epothilone PKS,the recombinant PKS produces an epothilone derivative in which thethiazole moiety of epothilone (or an epothilone derivative) is changedto an oxazole or 5-methyloxazole moiety, respectively. Alternatively,the present invention provides recombinant PKS enzymes composed of theproducts of the epoA, epoC, epoD, epoE, and epoF genes (or modifiedversions thereof) without an NRPS module or with an NRPS module from aheterologous PKS. The heterologous NRPS module can be expressed as adiscrete protein or as a fusion protein with either the epoA or epoCgenes.

In another embodiment, the invention provides recombinant epothilone PKSenzymes and corresponding recombinant DNA compounds and vectors in whichthe NRPS module has been inactivated or deleted. Inactive NRPS moduleproteins and the coding sequences therefore provided by the inventioninclude those in which the PCP domain has been wholly or partiallydeleted or otherwise rendered inactive by changing the active siteserine (the site for phosphopantetheinylation) to another amino acid,such as alanine, or the adenylation domains have been deleted orotherwise rendered inactive. In one embodiment, both the loading moduleand the NRPS have been deleted or rendered inactive. In any event, theresulting epothilone PKS can then function only if provided a substratethat binds to the KS domain of module 2 (or a subsequent module) of theepothilone PKS or a PKS for an epothilone derivative. In a methodprovided by the invention, the thus modified cells are then fedactivated acylthioesters that are bound by preferably the second, butpotentially any subsequent, module and processed into novel epothilonederivatives. The host cell is fed activated acylthioesters to producenovel epothilone derivatives of the invention. The host cellsexpressing, or cell free extracts containing, the PKS can be fed orsupplied with N-acylcysteamine thioesters (NACS) of novel precursormolecules to prepare epothilone derivatives. See U.S. patent applicationSer. No. 09/492,733, filed 27 Jan. 2000, and PCT patent publication No.US99/03986, both of which are incorporated herein by reference, andExamples 9 and 10, below.

The second (first non-NRPS) extender module of the epothilone PKSincludes a KS, an AT specific for methylmalonyl CoA, a DH, a KR, and anACP. The second extender module of the epothilone PKS is produced as adiscrete protein by the epoC gene. All or only a portion of the secondextender module coding sequence can be utilized in conjunction withother PKS coding sequences to create a hybrid module. In thisembodiment, the invention provides, for example, either replacing themethylmalonyl CoA specific AT with a malonyl CoA, ethylmalonyl CoA, or2-hydroxymalonyl CoA specific AT; deleting either the DH or KR or both;replacing the DH or KR or both with a DH or KR or both that specify adifferent stereochemistry; and/or inserting an ER. The resultingheterologous second extender module coding sequence can be coexpressedwith the other proteins that constitute a PKS that synthesizesepothilone, an epothilone derivative, or another polyketide.Alternatively, one can delete or replace the second extender module ofthe epothilone PKS with a module from a heterologous PKS, which can beexpressed as a discrete protein or as a fusion protein fused to eitherthe epoB or epoD gene product.

Illustrative recombinant PKS genes of the invention include those inwhich the AT domain encoding sequences for the second extender module ofthe epothilone PKS have been altered or replaced to change the AT domainencoded thereby from a methylmalonyl specific AT to a malonyl specificAT. Such malonyl specific. AT domain encoding nucleic acids can beisolated for example and without limitation, from the PKS genes encodingthe narbonolide PKS, the rapamycin PKS (i.e., modules 2 and 12), and theFK-520 PKS (i.e., modules 3, 7, and 8). When such a hybrid secondextender module is coexpressed with the other proteins constituting theepothilone PKS, the resulting epothilone derivative produced is a16-desmethyl epothilone. In a preferred embodiment, the hybrid PKS alsocontains a methylmalonyl CoA specific AT domain in extender module 4 andis expressed in a host cell lacking a functional epoK gene such that thecompound produced is 16-desmethyl epothilone D

In addition, the invention provides DNA compounds and vectors encodingrecombinant epothilone PKS enzymes and the corresponding recombinantproteins in which the KS domain of the second (or subsequent) extendermodule has been inactivated or deleted, as described in Example 9,below. In a preferred embodiment, this inactivation is accomplished bychanging the codon for the active site cysteine to an alanine codon. Aswith the corresponding variants described above for the NRPS module, theresulting recombinant epothilone PKS enzymes are unable to produce anepothilone or epothilone derivative unless supplied a precursor that canbe bound and extended by the remaining domains and modules of therecombinant PKS enzyme. Illustrative precursor compounds are describedin Example 10, below. Alternatively, one could simply provide suchprecursors to a host cell that expressed only the epoD, epoE, and epoFgenes.

The third extender module of the epothilone PKS includes a KS, an ATspecific for malonyl CoA, a KR, and an ACP. The third extender module ofthe epothilone PKS is expressed as a protein, the product of the epoDgene, which also contains modules 4; 5, and 6. To make a recombinantepothilone PKS that produces an epothilone derivative due to analteration in any of extender modules 3 through 6, one typicallyexpresses a protein comprising all four extender modules. In oneembodiment, all or a portion of the third extender module codingsequence is utilized in conjunction with other PKS coding sequences tocreate a hybrid module. In this embodiment, the invention provides, forexample, either replacing the malonyl CoA specific AT with amethylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specificAT; deleting the KR; replacing the KR with a KR that specifies adifferent stereochemistry; and/or inserting a DH or a DH and an ER. Theresulting heterologous third extender module coding sequence can beutilized in conjunction with a coding sequence for a PKS thatsynthesizes epothilone, an epothilone derivative, or another polyketide.

Illustrative recombinant PKS genes of the invention include those inwhich the AT domain encoding sequences for the third extender module ofthe epothilone PKS have been altered or replaced to change the AT domainencoded thereby from a malonyl specific AT to a methylmalonyl specificAT. Such methylmalonyl specific AT domain encoding nucleic acids can beisolated, for example and without limitation, from the PKS genesencoding DEBS, the narbonolide PKS, the rapamycin PKS, and the FK-520PKS. When coexpressed with the remaining modules and proteins of theepothilone PKS or an epothilone PKS derivative, the recombinant PKSproduces the 14-methyl epothilone derivatives of the invention.

Those of skill in the art will recognize that the KR domain of the thirdextender module of the PKS is responsible for forming the hydroxyl groupinvolved in cyclization of epothilone. Consequently, abolishing the KRdomain of the third extender module or adding a DH or DH and ER domainswill interfere with the cyclization, leading either to a linear moleculeor to a molecule cyclized at a different location than is epothilone.

The fourth extender module of the epothilone PKS includes a KS, an ATthat can bind either malonyl-CoA or methylmalonyl CoA, a KR, and an ACP.In one embodiment, all or a portion of the fourth extender module codingsequence is utilized in conjunction with other PKS coding sequences tocreate a hybrid module. In this embodiment, the invention provides, forexample, either replacing the malonyl CoA and methylmalonyl specific ATwith a malonyl CoA, methylmalonyl CoA, ethylmalonyl CoA, or2-hydroxymalonyl CoA specific AT; deleting the KR; and/or replacing theKR, including, optionally, to specify a different stereochemistry;and/or inserting a DH or a DH and ER. The resulting heterologous fourthextender module coding sequence is incorporated into a protein subunitof a recombinant PKS that synthesizes epothilone, an epothilonederivative, or another polyketide. Alternatively, the invention providesrecombinant PKS enzymes for epothilones and epothilone derivatives inwhich the entire fourth extender module has been deleted or replaced bya module from a heterologous PKS.

In a preferred embodiment, the invention provides recombinant DNAcompounds comprising the coding sequence for the fourth extender moduleof the epothilone PKS modified to encode an AT that binds methylmalonylCoA and not malonyl CoA. Thus, the invention provides recombinant DNAcompounds and expression vectors and the corresponding recombinant PKSin which the hybrid fourth extender module with a methylmalonyl specificAT has been incorporated. The methylmalonyl specific AT coding sequencecan originate, for example and without limitation, from coding sequencesfor the oleandolide PKS, DEBS, the narbonolide PKS, the rapamycin PKS,or any other PKS that comprises a methylmalonyl specific AT domain. Inaccordance with the invention, the hybrid fourth extender moduleexpressed from this coding sequence is incorporated into the epothilonePKS (or the PKS for an epothilone derivative), typically as a derivativeepoD gene product that comprises the modified fourth extender module aswell as extender modules 3, 5, and 6, any one or more of which canoptionally be in derivative form, of the epothilone PKS.

The recombinant methylmalonyl specific epothilone fourth extender modulecoding sequences provided by the invention afford important alternativemethods for producing desired epothilone compounds in host cells. Thus,the invention provides a hybrid fourth extender module coding sequencein which, in addition to the replacement of the endogenous AT codingsequence with a coding sequence for an AT specific for methylmalonyl CoA, coding sequences for a DH and KR for, for example and withoutlimitation, module 10 of the rapamycin PKS or modules 1 or 5 of theFK-520 PKS, have replaced the endogenous KR coding sequences. When thegene product comprising the hybrid fourth extender module and epothilonePKS modules 3, 5, and 6 (or derivatives thereof) encoded by this codingsequence is incorporated into a PKS comprising the other epothilone PKSproteins (or derivatives thereof) produced in a host cell, the cellmakes either epothilone D or its trans stereoisomer (or derivativesthereof, depending on the stereochemical specificity of the inserted DHand KR domains.

Similarly, and as noted above, the invention provides recombinant DNAcompounds comprising the coding sequence for the fourth extender moduleof the epothilone PKS modified to encode an AT that binds malonyl CoAand not methylmalonyl CoA. The invention provides recombinant DNAcompounds and vectors and the corresponding recombinant PKS in whichthis hybrid fourth extender module has been incorporated into aderivative epoD gene product. When incorporated into the epothilone PKS(or the PKS for an epothilone derivative), the resulting recombinantepothilone PKS produces epothilones C, A, and E, depending, again, onwhether epothilone modification enzymes are present. As noted above,depending on the host, whether the fourth extender module includes a KRand DH domain, and on whether and which of the dehydratase, epoxidase,and oxidase activities are present, the practitioner of the inventioncan produce one or more of the epothilone G, C, A, and E compounds andderivatives thereof using the compounds, host cells, and methods of theinvention.

In another embodiment, the present invention provides the13-oxo-epothilones by providing a recombinant epothilone PKS in whichthe KR domain of extender module 4 has been rendered inactive bymutation, delation, or replacement with a non-functional KR domain fromanother PKS. In a preferred embodiment, the invention provides arecombinant-host cell that produces only 13-oxo-epothilone D, becausethe recombinant PKS also has a replacement of the AT domain of module 4with a methylmalonyl specific AT domain, and no functional epoK gene ispresent in the cell. If the production of an epothilone derivativecompound is low due to an alteration in a module, production may beimproved by altering the KS and/or ACP domains of the succeeding module.

The fifth extender module of the epothilone PKS includes a KS, an ATthat binds malonyl CoA, a DH, an ER, a KR, and an ACP. In oneembodiment, a DNA compound comprising a sequence that encodes the fifthextender module of the epothilone PKS is inserted into a DNA compoundthat comprises coding sequences for the epothilone PKS or a recombinantepothilone PKS that produces an epothilone derivative. In anotherembodiment, a portion of the fifth extender module coding sequence isutilized in conjunction with other PKS coding sequences to create ahybrid module coding sequence and the hybrid module encoded thereby. Inthis embodiment, the invention provides, for example, either replacingthe malonyl CoA specific AT with a methylmalonyl CoA, ethylmalonyl CoA,or 2-hydroxymalonyl CoA specific AT; deleting any one, two, or all threeof the ER, DH, and KR; and/or replacing any one, two, or all three ofthe ER, DH, and KR with either a KR, a DH and KR, or a KR, DH, and ER,including, optionally, to specify a different stereochemistry. Theresulting hybrid fifth extender module coding sequence can be utilizedin conjunction with a coding sequence for a PKS that synthesizesepothilone, an epothilone derivative, or another polyketide.Alternatively, the fifth extender module of the epothilone PKS can bedeleted or replaced in its entirety by a module of a heterologous PKS toproduce a protein that in combination with the other proteins of theepothilone PKS or derivatives thereof constitutes a PKS that produces anepothilone derivative.

Illustrative recombinant PKS genes of the invention include recombinantepoD gene derivatives in which the AT domain encoding sequences for thefifth extender module of the epothilone PKS have been altered orreplaced to change the AT domain encoded thereby from a malonyl specificAT to a methylmalonyl specific AT. Such methylmalonyl specific AT domainencoding nucleic acids can be isolated, for example and withoutlimitation, from the PKS genes encoding DEBS, the narbonolide PKS, therapamycin PKS, and the FK-520 PKS. When such recombinant epoD genederivatives are coexpressed with the epoA, epoB, epoC, epoE, and epoFgenes (or derivatives thereof), the PKS composed thereof produces the10-methyl epothilones or derivatives thereof. Another recombinant epoDgene derivative provided by the invention includes not only this alteredmodule 5 coding sequence but also module 4 coding sequences that encodean AT domain that binds only methylmalonyl CoA. When incorporated into aPKS with the epoA, epoB, epoC, epoE, and epoF genes, the recombinantepoD gene derivative product leads to the production of 10-methylepothilone B and/or D derivatives.

Other illustrative recombinant epoD gene derivatives of the inventioninclude those in which the ER, DH, and KR domain encoding sequences forthe fifth extender module of the epothilone PKS have been: (i) replacedwith those encoding a KR and DH domain; (ii) either replaced with thoseencoding a KR domain or a KR domain and an inactive DH domain or fromwhich the DH domain coding sequence has been deleted or renderedinactive by mutation; and (iii) either replaced with those encoding aninactive KR domain or from which the KR domain coding sequence has beendeleted or rendered inactive by mutation. These recombinant epoD genederivatives of the invention are coexpressed with the epoA, epoB, epoC,epoE, and epoF genes to produce a recombinant PKS that makes thecorresponding (i) C-1 alkene, (ii) C-11 hydroxy (either epimer), and(iii) C-11 keto epothilone derivatives. These recombinant epoD genederivatives can also be coexpressed with recombinant epo genescontaining other alterations or can themselves be further altered toproduce a PKS that makes the corresponding C-11 epothilone derivatives.For example, one recombinant epoD gene derivative provided by theinvention also includes module 4 coding sequences that encode an ATdomain that binds only methylmalonyl CoA. When incorporated into a PKSwith the epoA, epoB, epoC, epoE, and epoF genes, the recombinant epoDgene derivative product leads to the production of the correspondingC-11 epothilone B and/or D derivatives, depending on whether afunctional epoK gene is present in the host cell.

As noted above, functionally similar epoD genes for producing theepothilone C-11 derivatives can also be made by inactivation of one,two, or all three of the ER, DH, and KR domains of the epothilone fifthextender module. However, the preferred mode for altering such domainsin any module is by replacement with the complete set of desired domainstaken from another module of the same or a heterologous PKS codingsequence. In this manner, the natural architecture of the PKS isconserved. Also, when present, KR and DH or KR, DH, and ER domains thatfunction together in a native PKS are preferably used in the recombinantPKS. Illustrative replacement domains for the substitutions describedabove include, for example and without limitation, the inactive KRdomain from the rapamycin PKS module 3 to form the ketone, the KR domainfrom the rapamycin PKS module 5 to form the alcohol, and the KR and DHdomains from the rapamycin PKS module 4 to form the alkene. Other suchinactive KR, active KR, and active KR and DH domain encoding nucleicacids can be isolated from, for example and without limitation, the PKSgenes encoding DEBS, the narbonolide PKS, and the FK-520 PKS. Each ofthe resulting PKS enzymes produces a polyketide compound that comprisesa functional group at the C-11 position that can be further derivatizedin vitro by standard chemical methodology to yield semi-syntheticepothilone derivatives of the invention.

The sixth extender module of the epothilone PKS includes a KS, an ATthat binds methylmalonyl CoA, a DH, an ER, a KR, and an ACP. In oneembodiment, a portion of the sixth extender module coding sequence isutilized in conjunction with other PKS coding sequences to create ahybrid module. In this embodiment, the invention provides, for example,either replacing the methylmalonyl CoA specific AT with a malonyl CoA,ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting any one,two, or all three of the ER, DH, and KR; and/or replacing any one, two,or all three of the ER, DH, and KR with either a KR, a DH and KR, or aKR, DH, and ER, including, optionally, to specify a differentstereochemistry. The resulting heterologous sixth extender module codingsequence can be utilized in conjunction with a coding sequence for aprotein subunit of a PKS that makes epothilone, an epothilonederivative, or another polyketide. Alternatively, the sixth extendermodule of the epothilone PKS can be deleted or replaced in its entiretyby a module from a heterologous PKS to produce a PKS for an epothilonederivative.

Illustrative recombinant PKS genes of the invention include those inwhich the AT domain encoding sequences for the sixth extender module ofthe epothilone PKS have been altered or replaced to change the AT domainencoded thereby from a methylmalonyl specific AT to a malonyl specificAT. Such malonyl specific AT domain encoding nucleic acids can beisolated from, for example and without limitation, the PKS genesencoding the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS.When a recombinant epoD gene of the invention encoding such a hybridmodule 6 is coexpressed with the other epothilone PKS genes, therecombinant PKS makes the 8-desmethyl epothilone derivatives. Thisrecombinant epoD gene derivative can also be coexpressed withrecombinant epo gene derivatives containing other alterations or canitself be further altered to produce a PKS that makes the corresponding8-desmethyl epothilone derivatives. For example, one recombinant epoDgene provided by the invention also includes module 4 coding sequencesthat encode an AT domain that binds only methylmalonyl CoA. Whenincorporated into a PKS with the epoA, epoB, epoC, epoE, and epoF genes,the recombinant epoD gene product leads to the production of the8-desmethyl derivatives of epothilones B and D.

Other illustrative recombinant epoD gene derivatives of the inventioninclude those in which the ER, DH, and KR domain encoding sequences forthe sixth extender module of the epothilone PKS have been replaced withthose that encode (i) a KR and DH domain; (ii) a KR domain; and (iii) aninactive KR domain. These recombinant epoD gene derivatives of theinvention, when coexpressed with the other epothilone PKS genes make thecorresponding (i) C-9 alkene, (ii) C-9 hydroxy (both epimers, only oneof which may be processed by downstream modules, unless additional KSand/or ACP replacements are made in the next module), and (iii) C-9 keto(C-9-oxo) epothilone derivatives. Functionally equivalent sixth extendermodules can also be made by inactivation of one, two, or all three ofthe ER, DH, and KR domains of the epothilone sixth extender module.These recombinant epoD gene derivatives can also be coexpressed withother recombinant epo gene derivatives containing other alterations orcan themselves be further altered to produce a PKS that makes thecorresponding C-9 epothilone derivatives. For example, one recombinantepoD gene derivative provided by the invention also includes module 4coding sequences that encode an AT domain that binds only methylmalonylCoA. When incorporated into a PKS with the epoA, epoB, epoC, epoE, andepoF genes, the recombinant epoD gene product leads to the production ofthe C-9 derivatives of epothilones B and D, depending on whether afunctional epoK gene is present.

Illustrative replacement domains for the substitutions described aboveinclude but are not limited to the inactive KR domain from the rapamycinPKS module 3 to form the ketone, the KR domain from the rapamycin PKSmodule 5 to form the alcohol, and the KR and DH domains from therapamycin PKS module 4 to form the alkene. Other such inactive KR,active KR, and active KR and DH domain encoding nucleic acids can beisolated from for example and without limitation the PKS genes encodingDEBS, the narbonolide PKS, and the FK-520 PKS. Each of the resultingPKSs produces a polyketide compound that comprises a functional group atthe C-9 position that can be further derivatized in vitro by standardchemical methodology to yield semi-synthetic epothilone derivatives ofthe invention.

The seventh extender module of the epothilone PKS includes a KS, an ATspecific for methylmalonyl CoA, a KR, and an ACP. The seventh extendermodule of the epothilone PKS is contained in the gene product of theepoE gene, which also contains the eighth extender module. In oneembodiment, a DNA compound comprising a sequence that encodes theseventh extender module of the epothilone PKS is expressed to form aprotein that, together with other proteins, constitutes the epothilonePKS or a PKS that produces an epothilone derivative. In these andrelated embodiments, the seventh and eighth extender modules of theepothilone PKS or a derivative thereof are typically expressed as asingle protein and coexpressed with the epoA, epoB, epoC, epoD, and epoFgenes or derivatives thereof to constitute the PKS. In anotherembodiment, a portion or all of the seventh extender module codingsequence is utilized in conjunction with other PKS coding sequences tocreate a hybrid module. In this embodiment, the invention provides, forexample, either replacing the methylmalonyl CoA specific AT with amalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT;deleting the KR; replacing the KR with a KR that specifies a differentstereochemistry; and/or inserting a DH or a DH and an ER. The resultingheterologous seventh extender module coding sequence is utilized,optionally in conjunction with other coding sequences, to express aprotein that together with other proteins constitutes a PKS thatsynthesizes epothilone, an epothilone derivative, or another polyketide.Alternatively, the coding sequences for the seventh extender module inthe epoE gene can be deleted or replaced by those for a heterologousmodule to prepare a recombinant epoE gene derivative that, together withthe epoA, epoB, epoC, epoD, and epoF genes, can be expressed to make aPKS for an epothilone derivative.

Illustrative recombinant epoE gene derivatives of the invention includethose in which the AT domain encoding sequences for the seventh extendermodule of the epothilone PKS have been altered or replaced to change theAT domain encoded thereby from a methylmalonyl specific AT to a malonylspecific AT. Such malonyl specific AT domain encoding nucleic acids canbe isolated from for example and without limitation the PKS genesencoding the narbonolide PKS, the rapamycin PKS, and the FK-520 PKS.When coexpressed with the other epothilone PKS genes, epQA, epoB, epoC,epoD, and epoF, or derivatives thereof, a PKS for an epothilonederivative with a C-6 hydrogen, instead of a C-6 methyl, is produced.Thus, if the genes contain no other alterations, the compounds producedare the 6-desmethyl epothilones.

The eighth extender module of the epothilone PKS includes a KS, an ATspecific for methylmalonyl CoA, inactive KR and DH domains, amethyltransferase (MT) domain, and an ACP. In one embodiment, a DNAcompound comprising a sequence that encodes the eighth extender moduleof the epothilone PKS is coexpressed with the other proteinsconstituting the epothilone PKS or a PKS that produces an epothilonederivative. In another embodiment, a portion or all of the eighthextender module coding sequence is utilized in conjunction with otherPKS coding sequences to create a hybrid module. In this embodiment, theinvention provides, for example, either replacing the methylmalonyl CoAspecific AT with a malonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonylCoA specific AT; deleting the inactive KR and/or the inactive DH;replacing the inactive KR and/or DH with an active KR and/or DH; and/orinserting an ER. The resulting heterologous eighth extender modulecoding sequence is expressed as a protein that is utilized inconjunction with the other proteins that constitute a PKS thatsynthesizes epothilone, an epothilone derivative, or another polyketide.Alternatively, the coding sequences for the eighth extender module inthe epoE gene can be deleted or replaced by those for a heterologousmodule to prepare a recombinant epoE gene that, together with the epoA,epoB, epoC, epoD, and epoF genes, can be expressed to make a PKS for anepothilone derivative.

The eighth extender module of the epothilone PKS also comprises amethylation or methyltransferase (MT) domain with an activity thatmethylates the epothilone precursor. This function can be deleted toproduce a recombinant epoD gene derivative of the invention, which canbe expressed with the other epothilone PKS genes or derivatives thereofthat makes an epothilone derivative that lacks one or both methylgroups, depending on whether the AT domain of the eighth extender modulehas been changed to a malonyl specific AT domain, at the correspondingC-4 position of the epothilone molecule.

The ninth extender module of the epothilone PKS includes a KS, an ATspecific for malonyl CoA, a KR, an inactive DH, and an ACP. The ninthextender module of the epothilone PKS is expressed as a protein, theproduct of the epoF gene, that also contains the TE domain of theepothilone PKS. In one embodiment a DNA compound comprising a sequencethat encodes the ninth extender module of the epothilone PKS isexpressed as a protein together with other proteins to constitute anepothilone PKS or a PKS that produces an epothilone derivative. In theseembodiments, the ninth extender module is typically expressed as aprotein that also contains the TE domain of either the epothilone PKS ora heterologous PKS. In another embodiment, a portion or all of the ninthextender module coding sequence is utilized in conjunction with otherPKS coding sequences to create a hybrid module. In this embodiment, theinvention provides, for example, either replacing the malonyl CoAspecific AT with a methylmalonyl CoA, ethylmalonyl CoA, or 2-hydroxymalonyl CoA specific AT; deleting the KR; replacing the KR with a KRthat specifies a different stereochemistry; and/or inserting a DH or aDH and an ER. The resulting heterologous ninth extender module codingsequence is coexpressed with the other proteins constituting a PKS thatsynthesizes epothilone, an epothilone derivative, or another polyketide.Alternatively, the present invention provides a PKS for an epothilone orepothilone derivative in which the ninth extender module has beenreplaced by a module from a heterologous PKS or has been deleted in itsentirety. In the latter embodiment, the TE domain is expressed as adiscrete protein or fused to the eighth extender module.

Illustrative examples of recombinant epothilone derivative PKS genes ofthe invention, which are identified by listing the altered specificitiesof the hybrid modules (the other modules having the same specificity asthe epothilone PKS), include:

(a) module 4 with methylmalonyl specific AT (mmAT) and a KR and module 2with a malonyl specific AT (mAT) and a KR;(b) module 4 with mmAT and module 3 with mmAT; (c) module 4 with mmATand module 5 with mmAT;(d) module 4 with mmAT and module 5 with mmAT and only a DH and KR;(e) module 4 with mmAT and module 5 with mmAT and only a KR;(f) module 4 with mmAT and module 5 with mmAT and only an inactive KR;(g) module 4 with mmAT and module 6 with mAT;(h) module 4 with mmAT and module 6 with mAT and only a DH and KR;(i) module 4 with mmAT and module 6 with mAT and only a KR;(j) module 4 with mmAT and module 6 with mAT and only an inactive KR;(k) module 4 with mmAT and module 7 with mAT;(l) hybrids (d) through (f), except that module 5 has an mAT;(m) hybrids (h) through (j) except that module 6 has an mmAT; and(n) hybrids (a) through (m) except that module 4 has an mAT.The above list is illustrative only and should not be construed aslimiting the invention, which includes other recombinant epothilone PKSgenes and enzymes with not only two hybrid modules other than thoseshown but also with three or more hybrid modules.

The host cells of the invention can be grown and fermented underconditions known in the art for other purposes to produce the compoundsof the invention. The compounds of the invention can be isolated fromthe fermentation broths of these cultured cells and purified by methodssuch as those in Example 3, below.

Thus, in another embodiment, the present invention provides novelepothilone derivative compounds in isolated and substantially pure formsuseful in agriculture, veterinary practice, and medicine. The termisolated refers to a compound or composition in a preparation that issubstantially free of contaminating or undesired materials or, withrespect to a compound or composition found in nature, substantially freeof the materials with which that compound or composition is associatedin its natural state. In one embodiment, the compounds are useful asfungicides. In another embodiment, the compounds are useful in cancerchemotherapy. In a preferred embodiment, the compound is an epothilonederivative that is at least as potent against tumor cells as epothiloneB or D. In another embodiment, the compounds are useful asimmunosuppressants. In another embodiment, the compounds are useful inthe manufacture of another compound. In a preferred embodiment, thecompounds are formulated in a mixture or solution for administration toa human or animal.

The novel epothilone analogs of the present invention, as well as theepothilones produced by the host cells of the invention, can bederivatized and formulated as described in PCT patent publication Nos.93/10121, 97/19086, 98/08849, 98/22461, 98/25929, 99/01124, 99/02514,99/07692, 99/27890, 99/39694, 99/40047, 99/42602, 99/43320, 99/43653,99/54318, 99/54319, 99/54330, 99/65913, 99/67252, 99/67253, and00/00485, and U.S. Pat. No. 5,969,145, each of which is incorporatedherein by reference.

Preferred compounds of the invention include the 14-methyl epothilonederivatives (made by utilization of the hybrid module 3 of the inventionthat has an AT that binds methylmalonyl CoA instead of malonyl CoA); the8,9-dehydro epothilone derivatives (made by utilization of the hybridmodule 6 of the invention that has a DH and KR instead of an ER, DH, andKR); the 10-methyl epothilone derivatives (made by utilization of thehybrid module 5 of the invention that has an AT that binds methylmalonylCoA instead of malonyl CoA); the 9-hydroxy epothilone derivatives (madeby utilization of the hybrid module 6 of the invention that has a KRinstead of an ER, DH, and KR); the 8-desmethyl-14-methyl epothilonederivatives (made by utilization of the hybrid module 3 of the inventionthat has an AT that binds methylmalonyl CoA instead of malonyl CoA and ahybrid module 6 that binds malonyl CoA instead of methylmalonyl CoA);and the 8-desmethyl-8,9-dehydro epothilone derivatives (made byutilization of the hybrid module 6 of the invention that has a DH and KRinstead of an ER, DH, and KR and an AT that specifies malonyl CoAinstead of methylmalonyl CoA). Illustrative examples of other preferrednovel epothilones of the invention that can be made using epothilonederivative PKS enzymes of the invention include9-oxo-11-hydroxy-epothilone D (module 4 AT replacement, module 6 KRinactivation, and module 5 DH inactivation); 9-hydroxy-11-oxo-epothiloneD (module 4 AT replacement, module 6 DH inactivation, and module 5 KRinactivation); and 9,11-dihydroxy-epothilone D (module 4 AT replacement,module 6 DH inactivation, and module 5 DH inactivation).

The compounds of the invention can be readily formulated to provide thepharmaceutical compositions of the invention. The pharmaceuticalcompositions of the invention can be used in the form of apharmaceutical preparation, for example, in solid, semisolid, or liquidform. This preparation will contain one or more of the compounds of theinvention as an active ingredient in admixture with an organic orinorganic carrier or excipient suitable for external, enteral, orparenteral application. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, pessaries, solutions,emulsions, suspensions, and any other form suitable for use.

The carriers which can be used include water, glucose, lactose, gumacacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc,corn starch, keratin, colloidal silica, potato starch, urea, and othercarriers suitable for use in manufacturing preparations, in solid,semi-solid, or liquified form. In addition, auxiliary stabilizing,thickening, and coloring agents and perfumes may be used. For example,the compounds of the invention may be utilized with hydroxypropylmethylcellulose essentially as described in U.S. Pat. No. 4,916,138,incorporated herein by reference, or with a surfactant essentially asdescribed in EPO patent publication No. 428,169, incorporated herein byreference.

Oral dosage forms may be prepared essentially as described by Hondo etal., 1987, Transplantation Proceedings XIX, Supp. 6: 17-22, incorporatedherein by reference. Dosage forms for external application may beprepared essentially as described in EP Pub. No. 423,714, incorporatedherein by reference. The active compound is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the disease process or condition.

For the treatment of conditions and diseases caused by infection, immunesystem disorder (or to suppress immune function), or cancer, a compoundof the invention may be administered orally, topically, parenterally, byinhalation spray, or rectally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvant,and vehicles. The term parenteral, as used herein, includes subcutaneousinjections, and intravenous, intrathecal, intramuscular, andintrasternal injection or infusion techniques.

Dosage levels of the compounds of the present invention are of the orderfrom about 0.01 mg to about 100 mg per kilogram of body weight per day,preferably from about 0.1 mg to about 50 mg per kilogram of body weightper day. The dosage levels are useful in the treatment of theabove-indicated conditions (from about 0.7 mg to about 3.5 mg perpatient per day, assuming a 70 kg patient). In addition, the compoundsof the present invention may be administered on an intermittent basis,i.e., at semi-weekly, weekly, semi-monthly, or monthly intervals.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated sand the particular mode of administration. For example, aformulation intended for oral administration to humans may contain from0.5 mg to 5 gm of active agent compounded with an appropriate andconvenient amount of carrier material, which may vary from about 5percent to about 95 percent of the total composition. Dosage unit formswill generally contain from about 0.5 mg to about 500 mg of activeingredient. For external administration, the compounds of the inventionmay be formulated within the range of, for example, 0.00001% to 60% byweight, preferably from 0.001% to 10% by weight, and most preferablyfrom about 0.005% to 0.8% by weight.

It will be understood, however, that the specific dose level for anyparticular patient will depend on a variety of factors. These factorsinclude the activity of the specific compound employed; the age, bodyweight, general health, sex, and diet of the subject; the time and routeof administration and the rate of excretion of the drug; whether a drugcombination is employed in the treatment; and the severity of theparticular disease or condition for which therapy is sought.

In another embodiment, the present invention provides a method oftreating cancer, which method comprises administering a therapeuticallyeffective amount of a novel epothilone compound of the invention.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating the presentinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

EXAMPLE 1 Construction of a Myxococcus xanthus Expression Vector

The DNA providing the integration and attachment function of phage Mx8was inserted into commercially available pACYC184 (New England Biolabs).An ˜2360 bp MfeI-SmaI from plasmid pPLH343, described in Salmi et al.,February 1998, J. Bact. 180(3): 614-621, was isolated and ligated to thelarge EcoRI-XmnI restriction fragment of plasmid pACYC184. The circularDNA thus formed was ˜6 kb in size and called plasmid pKOS35-77.

Plasmid pKOS35-77 serves as a convenient plasmid for expressingrecombinant PKS genes of the invention under the control of theepothilone PKS gene promoter. In one illustrative embodiment, the entireepothilone PKS gene with its homologous promoter is inserted in one ormore fragments into the plasmid to yield an expression vector of theinvention.

The present invention also provides expression vectors in which therecombinant PKS genes of the invention are under the control of aMyxococcus xanthus promoter. To construct an illustrative vector, thepromoter of the pilA gene of M. xanthus was isolated as a PCRamplification product. Plasmid pSWU357, which comprises the pilA genepromoter and is described in Wu and Kaiser, December 1997, J. Bact.179(24):7748-7758, was mixed with PCR primers Seq1 and Mxpil1 primers:

Seq1: 5′-AGCGGATAACAATTTCACACAGGAAACAGC-3′; and Mxpil1:5′-TTAATTAAGAGAAGGTTGCAACGGGGGGC-3′,and amplified using standard PCR conditions to yield an ˜800 bpfragment. This fragment was cleaved with restriction enzyme KpnI andligated to the large KpnI-EcoRV restriction fragment of commerciallyavailable plasmid pLitmus 28 (New England Biolabs). The resultingcircular DNA was designated plasmid pKOS35-71B.

The promoter of the pilA gene from plasmid pKOS35-71B was isolated as an˜800 bp EcoRV-SnaBI restriction fragment and ligated with the large MscIrestriction fragment of plasmid pKOS35-77 to yield a circular DNA ˜6.8kb in size. Because the ˜800 bp fragment could be inserted in either oneof two orientations, the ligation produced two plasmids of the samesize, which were designated as plasmids pKOS35-82.1 and pKOS35-82.2.Restriction site and function maps of these plasmids are presented inFIG. 2.

Plasmids pKOS35-82.1 and pKOS35-82.2 serve as convenient startingmaterials for the vectors of the invention in which a recombinant PKSgene is placed under the control of the Myxococcus xanthus pilA genepromoter. These plasmids comprise a single Pad restriction enzymerecognition sequence placed immediately downstream of the transcriptionstart site of the promoter. In one illustrative embodiment, the entireepothilone PKS gene without its homologous promoter is inserted in oneor more fragments into the plasmids at the Pad site to yield expressionvectors of the invention.

The sequence of the pilA promoter in these plasmids is shown below.

CGACGCAGGTGAAGCTGCTTCGTGTGCTCCAGGAGCGGAAGGTGAAGCCGGTCGGCAGCGCCGCGGAGATTCCCTTCCAGGCGCGTGTCATCGCGGCAACGAACCGGCGGCTCGAAGCCGAAGTAAAGGCCGGACGCTTTCGTGAGGACCTCTTCTACCGGCTAACGTCATCACGTTGGAGCTGCCTCCACTGCGCGAGCGTTCCGGCGACGTGTCGTTGCTGGCGAACTACTTCCTGTCCAGACTGTCGGAGGAGTTGGGGCGACCCGGTCTGCGTTTCTCCCCCGAGACACTGGGGCTATTGGAGCGCTATCCCTTCCCAGGCAACGTGCGGCAGCTGCAGAACATGGTGGAGCGGGCCGCGACCCTGTCGGATTCAGACCTCCTGGGGCCCTCCACGCTTCCACCCGCAGTGCGGGGCGATACAGACCCCGCCGTGCGTCCCGTGGAGGGCAGTGAGCCAGGGCTGGTGGCGGCTTCAACCTGGAGCGGCATCTCGACGACAGCGAGCGGCGCTATCTCGTCGCGGCGATGAAGCAGGCCGGGGGCGTGAAGACCCGTGCTGCGGAGTTGCTGGGCCTTTCGTTCCGTTCATTCCGCTACCGGTTGGCCAAGCATGGGCTGACGGATGACTTGGAGCCCGGGAGCGCTTCGGATGCGTAGGCTGATCGACAGTTATCGTCAGCGTCACTGCCGAATTTTGTCAGCCCTGGACCCATCCTCGCCGAGGGGATTGTTCCAAGCCTTGAGAATTGGGGGGCTTGGAGTGCGCACCTGGGTTGGCATGCGTAGTGCTAATCCCATCCGCGGGCGCAGTGCCCCCCGTTGCAACCTTCTCTTAATTAA

To make the recombinant Myxococcus xanthus host cells of the invention,M. xanthus cells are grown in CYE media (Campos and Zusman, 1975,Regulation of development in Myxococcus xanthus: effect of 3′:5′-cyclicAMP, ADP, and nutrition, Proc. Natl. Acad. Sci. USA 72: 518-522) to aKlett of 100 at 30° C. at 300 rpm. The remainder of the protocol isconducted at 25° C. unless otherwise indicated. The cells are thenpelleted by centrifugation (8000 rpm for 10 min. in an SS34 or SA600rotor) and resuspended in deionized water. The cells are again pelletedand resuspended in 1/100th of the original volume.

DNA (one to two μL) is electroporated into the cells in a 0.1 cm cuvetteat room temperature at 400 ohm, 25 μFD, 0.65 V with a time constant inthe range of 8.8-9.4. The DNA is free of salts and is resuspended indistilled and deionized water or dialyzed on a 0.025 μm Type VS membrane(Millipore). For low efficiency electroporations, the DNA is spotdialyzed, and outgrowth is in CYE. Immediately after electroporation, 1mL of CYE is added, and the cells in the cuvette pooled with anadditional 1.5 mL of CYE previously added to a 50 mL Erlenmeyer flask(total volume 2.5 ml). The cells are grown for four to eight hours (orovernight) at 30 to 32° C. at 300 rpm to allow for expression of theselectable marker. Then, the cells are plated in CYE soft agar on plateswith selection. With kanamycin as the selectable marker, typical yieldsare 10³ to 10⁵ per μg of DNA. With streptomycin as the selectablemarker, it is included in the top agar, because it binds agar.

With this procedure, the recombinant DNA expression vectors of theinvention are electroporated into Myxococcus host cells that expressrecombinant PKSs of the invention and produce the epothilone, epothilonederivatives, and other novel polyketides encoded thereby.

EXAMPLE 2 Construction of a Bacterial Artificial-Chromosome (BAC) forExpression of Epothilone in Myxococcus xanthus

To express the epothilone PKS and modification enzyme genes in aheterologous host to produce epothilones by fermentation, Myxococcusxanthus, which is closely related to Sorangium cellulosum and for whicha number of cloning vectors are available, is employed in accordancewith the methods of the invention. M. xanthus and S. cellulosum aremyxobacteria and so may share common elements of gene expression,translational control, and post translational modification (if any). M.xanthus has been developed for gene cloning and expression: DNA can beintroduced by electroporation, and a number of vectors and geneticmarkers are available for the introduction of foreign DNA, includingthose that permit its stable insertion into the chromosome. M. xanthuscan be grown with relative ease in complex media in fermentors and canbe subjected to manipulations to increase gene expression, if required.

To introduce the epothilone gene cluster into Myxococcus xanthus, onecan build the epothilone cluster into the chromosome by using homologousrecombination to assemble the complete gene cluster. Alternatively, thecomplete epothilone gene cluster can be cloned on a bacterial artificialchromosome (BAC) and then moved into M. xanthus for integration into thechromosome.

To assemble the gene cluster from cosmids pKOS35-70.1A2, andpKOS35-79.85, small regions (˜2 kb or larger) of homology from thesecosmids are introduced into Myxococcus xanthus to provide recombinationsites for larger pieces of the gene cluster. As shown in FIG. 3,plasmids pKOS35-154 and pKOS90-22 are created to introduce theserecombination sites. The strategy for assembling the epothilone genecluster in the M. xanthus chromosome is shown in FIG. 4. Initially, aneutral site in the bacterial chromosome is chosen that does not disruptany genes or transcriptional units. One such region is downstream of thedevS gene, which has been shown not to affect the growth or developmentof M. xanthus. The first plasmid, pKOS35-154, is linearized with DraIand electroporated into M. xanthus. This plasmid contains two regions ofthe dev locus flanking two fragments of the epothilone gene cluster.Inserted in between the epo gene regions is a cassette composed of akanamycin resistance marker and the E. coli galK gene. See Ueki et al.,1996, Gene 183: 153-157, incorporated herein by reference. Kanamycinresistance arises in colonies if the DNA recombines into the dev regionby a double recombination using the dev sequence as regions of homology.

This strain, K35-159, contains small (˜2.5 kb) regions of the epothilonegene cluster that will allow for recombination of pKOS35-79.85. Becausethe resistance markers on pKOS35-79.85 are the same as that in K35-159,a tetracycline transposon was transposed into the cosmid, and cosmidsthat contain the transposon inserted into the kanamycin marker wereselected. This cosmid, pKOS90-23, was electroporated into K35459, andoxytetracycline resistant colonies were selected to create strainK35-174. To remove the unwanted regions from the cosmid and leave onlythe epothilone genes, cells were plated on CYE plates containing 1%galactose. The presence of the galK gene makes the cells sensitive to 1%galactose. Galactose resistant colonies of K35474 represent cells thathave lost the galK marker by recombination or by a mutation in the galKgene. If the recombination event occurs, then the galactose resistantstrain is sensitive to kanamycin and oxytetracycline. Strains sensitiveto both antibiotics are verified by Southern blot analysis. The correctstrain is identified and designated K35-175 and contains the epothilonegene cluster from module 7 to 4680 bp downstream of the stop codon ofepoK.

To introduce modules 1 through module 7, the above process is repeatedonce more. The plasmid pKOS90-22 is linearized with DraI andelectroporated into K35475 to create K111-13.2. This strain iselectroporated with the tetracycline resistant version of pKOS35-70.1A2,pKOS90-38, and colonies resistant to oxytetracycline are selected. Thiscreates strain K111-13.23. Recombinants that now have the wholeepothilone gene cluster are selected by resistance to 1% galactose. Thisresults in clones K111-32.25, K111-32.26, and K111-32.35. StrainK111-32.25 has been deposited with the ATCC in compliance with theBudapest Treaty and is available under accession No. ATCC ______. Thisstrain contains all the epothilone genes and their promoters.

Fermentation was performed by inoculating strains into 5 mL of CYE in a50 mL flask and growing overnight until the culture was in mid loggrowth phase. A 100 μL aliquot was spread onto a CTS plate, and theplate incubated at 32° C. for 4 to 5 days. To extract epothilones, theagar plate with cells was macerated, put in a 50 mL conical tube, andacetone added to fill the tube. The solution was incubated with rockingfor 4 to 5 hours, the acetone evaporated, and the remaining liquidextracted twice with an equal volume of ethyl acetate. The water wasremoved from the ethyl acetate extract by adding magnesium sulfate. Themagnesium sulfate was filtered out, and the liquid was evaporated todryness. The epothilones were resuspended in 200 μL of acetonitrile andanalyzed by LC/MS. The analysis showed that the strain producedepothilones A and B, with epothilone B present at about 0.5 mg/L in theculture, and epothilone A at 5 to 10-fold lower levels.

Alternatively, this strain can be used to produce epothilones in liquidculture. A flask containing CYE is inoculated with an epothiloneproducing strain. The next day, while the cells are in mid-log growthphase, a 5% inoculum is added to a flask containing 0.5% CMM (0.5%casitone, 0.2% MgSO4.7H2O, 10 mM MOPS pH7.6) along with 1 mg/ml serine,alanine, and glycine and 0.1% sodium pyruvate. The sodium pyruvate canbe added to 0.5% to increase epothilone B production but causes adecrease in the ratio of epothilone B to epothilone A. The culture isgrown at 30° C. for 60-72 hours. Longer incubations result in a decreasein titers of epothilones. To recover epothilones, the cultures arecentrifuged at 10,000 rpm for 10 minutes in an SS34 rotor. Thesupernatants are extracted twice with ethyl acetate and rotavaped todryness. Liquid cultures produced 2 to 3 mg/L of epothilones A and B,with ratios similar to that observed with plate cultures. If XAD(0.5-2%) was added to the culture, epothilones C and D were observed,with epothilone D present at 0.5 to 1 mg/L and epothilone C present at 5to 10-fold lower levels.

To clone the whole gene cluster as one fragment, a bacterial artificialchromosome (BAC) library is constructed. First, SMP44 cells are embeddedin agarose and lysed according to the BIO-RAD genomic DNA plug kit. DNAplugs are partially digested with restriction enzyme, such as Sau3AI orHindIII, and electrophoresed on a FIGE or CHEF gel. DNA fragments areisolated by electroeluting the DNA from the agarose or using gelase todegrade the agarose. The method of choice to isolate the fragments iselectroelution, as described in Strong et al., 1997, Nucleic Acids Res.19: 3959-3961, incorporated herein by reference. The DNA is ligated intothe BAC (pBeloBACII) cleaved with the appropriate enzyme. A map ofpBeloBACII is shown below.

The DNA is electroporated into DH10B cells by the method of Sheng etal., 1995, Nucleic Acids Res. 23: 1990-1996, incorporated herein byreference, to create a Sorangium cellulosum genomic library. Coloniesare screened using a probe from the NRPS region of the epothilonecluster. Positive clones are picked and DNA is isolated for restrictionanalysis to confirm the presence of the complete gene cluster. Thispositive clone is designated pKOS35-178.

To create a strain that can be used to introduce pKOS35-178, a plasmid,pKOS35-164, is constructed that contains regions of homology that areupstream and downstream of the epothilone gene cluster flanked by thedev locus and containing the kanamycin resistance galK cassette,analogous to plasmids pKOS90-22 and pKOS35-154. This plasmid islinearized with DraI and electroporated into Myxococcus xanthus, inaccordance with the method of Kafeshi et al., 1995, Mol. Microbiol. 15:483-494, to create K35-183. The plasmid pKOS35-178 can be introducedinto K35-183 by electroporation or by transduction with bacteriophageP1, and chloramphenicol resistant colonies are selected. Alternatively,a version of pKOS35-178 that contains the origin of conjugative transferfrom pRP4 can be constructed for transfer of DNA from E. coli toK35-183. This plasmid is made by first constructing a transposoncontaining the oriT region from RP4 and the tetracycline resistancemaker from pACYC184 and then transposing the transposon in vitro or invivo onto pKOS35-178. This plasmid is transformed into S17-1 andconjugated into M. xanthus. This strain, K35-190, is grown in thepresence of 1% galactose to select for the second recombination event.This strain contains all the epothilone genes as well as all potentialpromoters. This strain is fermented and tested for the production ofepothilones A and B.

Alternatively, the transposon can be recombined into the BAC usingeither the temperature sensitive plasmid pMAK705 or pKO3 by transposingthe transposon onto either pMAK705 or pKO3, selecting for tetR and camSplasmids; the recombination is accomplished as described in Hamilton etal., September 1989, J. Bact. 171(9): 4617-4622 and Link et al., October1997, J. Bact. 179(20): 6228-6237, each of which is incorporated hereinby reference.

Besides integrating pKOS35-178 into the dev locus, it can also beintegrated into a phage attachment site using integration functions frommyxophages Mx8 or Mx9. A transposon is constructed that contains theintegration genes and att site from either Mx8 or Mx9 along with thetetracycline gene from pACYC184. Alternative versions of this transposonmay have only the attachment site. In this version, the integrationgenes are then supplied in trans by coelectroporation of a plasmidcontaining the integrase gene or having the integrase protein expressedin the electroporated strain from any constitutive promoter, such as themgl promoter (see Magrini et al., July 1999, J. Bact. 181(13):4062-4070, incorporated herein by reference). Once the transposon isconstructed, it is transposed onto pKOS35-178 to create pKOS35-191. Thisplasmid is introduced into Myxococcus xanthus as described above. Thisstrain contains all the epothilone genes as well as all potentialpromoters. This strain is fermented and tested for the production ofepothilones A and B.

Once the epothilone genes have been established in a strain ofMyxococcus xanthus, manipulation of any part of the gene cluster, suchas changing promoters or swapping modules, can be performed using thekanamycin resistance and galK cassette, as described below. Cultures ofMyxococcus xanthus containing the epo genes are grown in a number ofmedia and examined for production of epothilones. If the levels ofproduction of epothilones (in particular B or D) are low, then the M.xanthus-producing clones are subjected to media development and mutationbased strain improvement.

EXAMPLE 3 Process for the Production of Epothilones B and D

A. Production of epothilone B

I. Flasks

A 1 mL vial of the K111-32-25 strain is thawed and the contentstransferred into 3 mL of CYE seed media in a glass tube. This culture isincubated for 72±12 hours at 30° C., followed by the subculturing of 3mL of this tube culture into 50 mL of CYE media within a 250 mL baffledErlenmeyer flask. This CYE flask is incubated for 24±8 hours at 30° C.,and 2.5 mL of this seed (5% v/v) used to inoculate the epothiloneproduction flasks (50 mL of CTS-TA media in a 250 mL baffled Erlenmeyerflask). These flasks are then incubated at 30° C. for 48±12 hours, witha media pH at the beginning of 7.4. The peak epothilone A titer is 0.5mg/t, and the peak epothilone B titer is 2.5 mg/L.

II. Fermentors

A similar inoculum expansion of K111-32-25 as described above is used,with the additional step that 25 mL of the 50 mL CYE seed is subculturedinto 500 Mt of CYE. This secondary seed is used to inoculate a 10 Lfermentor containing 9.5 L of CTS-TA, and 1 g/L of sodium pyruvate. Theprocess parameter setpoints for this fermentation are: pH—7.4;agitation—400 rpm; sparge rate—0.15 vvm. These parameters weresufficient to maintain the DO at greater than 80% of saturation. The pHcontrol is provided by addition of 2.5 N sulfuric acid and sodiumhydroxide to the cultures. Peak epothilone titers are achieved at 48±8hours. The peak epothilone A titer is 1.6 mg/L, and the peak epothiloneB titer is 5.2 mg/L.

B. Production of Epothilone D

I. Flasks

A 1 mL vial of the K111-40-1 strain (described in a following example)is thawed and the contents transferred into 3 mL of CYE seed media in aglass tube. This culture is incubated for 72±12 hours at 30° C.,followed by the subculturing of 3 mL of this tube culture into 50 mL ofCYE media within a 250 mL baffled Erlenmeyer flask. This CYE flask isincubated for 24±8 hours at 30° C., and 2.5 mL of this seed (5% v/v)used to inoculate the epothilone production flasks (50 mL of 1× wheatgluten media in a 250 mL baffled Erlenmeyer flask). These flasks arethen incubated at 30° C. for 48±12 hours, with a media pH at thebeginning of 7.4. The peak epothilone C titer is 1.4 mg/L, and the peakepothilone D titer is 7.2 mg/L.

II. Fermentors

A similar inoculum expansion of K111-40-1 as described above is used,with the additional step that 25 mL of the 50 mL CYE seed is subculturedinto 500 mL of CYE. 250 mL of this secondary seed is used to inoculate a5 L fermentor containing 4.5 L of CIS-TA, with a 1 g/L daily feed ofsodium pyruvate. The process parameter setpoints for this fermentationare: pH—7.4; agitation—400 rpm; sparge rate—0.15 vvm. These parameterswere sufficient to maintain the DO at greater than 80% of saturation.The pH control is provided by addition of 2.5 N sulfuric acid and sodiumhydroxide to the cultures. Peak epothilone titers are achieved at 36±8hours. The peak epothilone C titer is 0.5 mg/L, and the peak epothiloneD titer is 1.6 mg/L.

CYE Seed Media Component Concentration Casitone (Difco) 10 g/L YeastExtract (Difco) 5 g/L MgSO₄_7H₂0 (EM Science) 1 g/L HEPES buffer 50 mMSterilized by autoclaving for 30 minutes at 121° C.

CTS-TA Production Media Component Concentration Casitone (Difco) 5 g/LMgSO₄_7H₂0 (EM Science) 2 g/L L-alanine, L-serine, glycine 1 mg/L HEPESbuffer 50 mMSterilized by autoclaving for 30 minutes at 121° C.

1x Wheat Gluten Production Media Component Concentration Wheat Gluten(Sigma) 5 g/L MgSO₄_7H₂0 (EM Science) 2 g/L HEPES buffer 50 mMSterilized by autoclaving for 45 minutes at 121° C.

EXAMPLE 4 Construction of a Myxococcus Strain with Non-Functional epoKGene

Strain K111-40-1 was constructed from strain K111-32.25 by insertionalinactivation of the epoK gene. To construct an epoK mutant, a kanamycinresistance cassette was inserted into the epoK gene. This was done byisolating the 4879 bp fragment from pKOS35-79.85, which contains epoK,and ligating it into the NotI site of pBluescriptSKII+. This plasmid,pKOS35-83.5, was partially cleaved with ScaI, and the 7.4 kb fragmentwas ligated with the 1.5 kb EcoRI-BamHI fragment containing thekanamycin resistance gene from pBJ180-2, which had the DNA ends madeblunt with the Klenow fragment of DNA polymerase I, to yield plasmidpKOS90-55. Finally, the ˜400 bp RP4 oriT fragment from pBJ183 wasligated into the XbaI and EcoRI sites to create pKOS90-63. This plasmidwas linearized with DraI and electroporated into the Myxococcus xanthusstrain K111-32.25.

To create a markerless epoK mutation, pKOS35-83.5 was cleaved with ScaIand the 2.9 kb and 4.3 kb fragments were ligated together. This plasmid,pKOS90-101, has an in-frame deletion in epoK. Next, the 3 kb BamHI andNdeI fragment from KG2, which had the DNA ends made blunt with theKlenow fragment of polymerase I and contains the kanamycin resistanceand galK genes, was ligated into the DraI site of pKOS90-101 to createpKOS90-105. This plasmid was electroporated into K111-32.25 andkanamycin resistant electroporants were selected. To replace the wildtype copy of epoK with the deletion, the second recombination event wasselected by growth on galactose plates. These galactose resistantcolonies are tested for production of epothilone C and D, and aproducing strain is designated K111-72.

EXAMPLE 5 Addition of matBC

The matBC genes encode a malonyl-CoA synthetase and a dicarboxylatecarrier protein, respectively. See An and Kim, 1998, Eur. J. Biochem.257: 395-402, incorporated herein by reference. These two proteins areresponsible for the conversion of exogenous malonate to malonyl-CoAinside the cell. The products of the two genes can transportdicarboxylic acids and convert them to CoA derivatives (see U.S. patentapplication Ser. No. 60/159,060, filed 13 Oct. 1999, incorporated hereinby reference). These two genes can be inserted into the chromosome ofMyxococcus xanthus to increase the cellular concentrations ofmalonyl-CoA and methymalonyl-CoA to increase polyketide production. Thisis accomplished by cleaving pMATOP-2 with BglII and SpeI and ligating itinto the BglII and SpeI sites of pKOS35-82.1, which contains thetetracycline resistance conferring gene, the Mx8 att site and the M.xanthus pilA promoter to drive expression of matBC. This plasmid can beelectroporated into M. xanthus. Because the pilA promoter is highlytranscribed, it may be necessary to insert a weaker promoter in theevent that too much MatB and MatC affect cell growth. Alternativepromoters include the promoter of the kanamycin resistance conferringgene.

EXAMPLE 6 Mutation of the KS^(Y) in the Loading Module

The proposed mechanism of initiation of epothilone biosynthesis is thebinding of malonate to the ACP of the loading domain and the subsequentdecarboxylation by the loading KS domain. The loading KS domain containsa tyrosine at the active site cysteine (KS^(Y)) which renders it unableto perform the condensation reaction. However, it is proposed to stillperform the decarboxylation. Recent work with rat fatty acid synthasehas shown that a KS domain which contains a glutamine in the active sitecysteine (KS^(Q)) increases the decarboxylation by two orders ofmagnitude whereas changing this amino acid to serine, alanine,asparagine, glycine or threonine resulted in no increase relative towild type. Therefore, changing the KS^(Y) to KS^(Q) may increase thepriming of epothilone resulting in an increase in epothilone production.To make the change in strain K111-32.25, the plasmid pKOS39-148 wasconstructed that has ˜850 bp of the epothilone KS loading module codingsequence. The KS^(Q) mutation was created in this plasmid by sitedirected mutagenesis. To perform a gene replacement in K111-32.25, thekanamycin resistance and galK genes from KG2 were inserted into the DraIsites of pKOS39-148 to create plasmids pKOS111-56.2A and pKOS111-56.2B.The plasmids differ in their orientation of the kanamycin-galK cassette.These plasmids were electroporated into K111-32.25 and kanamycinresistant colonies were selected to create strains K111-63. To replacethe wild type loading module KS, K111-63 was plated on CYE galactoseplates, and colonies were screened for the presence of the KSQ mutationby PCR and sequencing.

EXAMPLE 7 Addition of mtaA

To increase the levels of P pant transferase protein, the Ppanttransferase from Stigmatella aurantiaca strain DW4 can be added toK111-32.25. This is done by PCR amplification of mtaA from DW4chromosomal DNA using the primers 111-44.1(AAAAGCTTCGGGGCACCTCCTGGCTGTCGGC) and 111-44.4(GGTTAATTAATCACCCTCCTCCCACCCCGGGCAT). See Silakowski et al., 1999, J.Biol. Chem. 274(52):37391-37399, incorporated herein by reference. The˜800 bp fragment was cleaved with NcoI and ligated into the pUHE24-2Bthat had been cleaved with PstI, the DNA ends made blunt with the Klenowfragment of DNA polymerase I, and cleaved with NcoI. This plasmid isdesignated pKOS111-54. The mtaA gene is transferred to plasmidpKOS35-82.1, which contains the tetracycline resistance conferring gene,the Mx8 att site and the Myxococcus xanthus pilA promoter to driveexpression of mtaA. This plasmid is introduced into M. xanthus andintegrated into the Mx8 phage attachment site.

EXAMPLE 8 Construction of Promoter Replacement Plasmids

To improve epothilone production levels and to illustrate the widevariety of promoters that can be used to express PKS genes in host cellsof the invention, a series of vectors and host cells can be constructedto replace the Sorangium cellulosum epothilone PKS gene promoter withother suitable promoters, as described in this example.

A. Construction of Plasmid with Downstream Flanking Region

Cosmid pKOS35-70.8A3 was cut with NsiI and AvrII. The 9.5 kb fragmentwas ligated with pSL1190 cut with PstI and AvrII to yield pKOS90-13.Plasmid pKOS90-13 is ˜12.9 kb. Plasmid pKOS90-13 was cut withEcoRI/AvrII. The 5.1 kb fragment was ligated with pBluescript digestedwith EcoRI/SpeI to create pKOS90-64 (˜8.1 kb). This plasmid contains thedownstream flanking region for the promoter (epoA and some sequenceupstream of the start codon). The EcoRI site is ˜220 bp upstream fromthe start codon for the epoA gene. The AvrII site is 5100 bp downstreamfrom the EcoRI site.

B. Cloning of Upstream Flanking Region

Primers 90-66.1 and 90-67 (shown below) were used to clone the upstreamflanking region. Primer 90-67 is at the 5′ end of the PCR fragment and90-66.1 is at the 3′ end of the PCR fragment. The fragment ends 2481 bpbefore the start codon for the epoA gene. The ˜2.2 kb fragment was cutwith HindIII. Klenow polymerase was added to blunt the HindIII site.This fragment was ligated into the HincII site of pNEB193. Clones withthe proper orientation, those with the EcoRI site at the downstream endof the insert and HindIII at the upstream end of the insert, wereselected and named pKOS90-90.

C. Construction of Final Plasmid

Plasmid pKOS90-90 was cut with EcoRI and HindIII. The 2.2 kb fragmentwas ligated with pKOS90-64 digested with EcoRI/HindIII to create pKOS90-91 (10.3 kb). Plasmid pKOS90-91 contains the upstream flanking regionof the promoter followed by the downstream flanking region inpBluescript. There is a Pad site between the two flanking regions toclone promoters of interest. The galK/kan^(r) cassette was then insertedto enable recombination into Myxococcus xanthus. Plasmid pKOS90-91 wascut with DraI. DraI cuts once in the amp gene and twice in the vector(near the amp gene). Plasmid KG-2 was cut with BamHI/NdeI and Klenowpolymerase added to blunt the fragment. The 3 kb fragment (containinggalK/kan^(r) genes) was ligated with the ˜9.8 kb DraI fragment ofpKOS90-91 to create pKOS90-102 (12.8 kb).

D. Construction of Plasmid with Alternative Leader

The native leader region of the epothilone PKS genes can be replaced aleader with a different ribosome binding site. Plasmid pKOS39-136(described in U.S. patent application Ser. No. 09/443,501, filed 19 Nov.1999) was cut with PacI/AscI. The 3 kb fragment containing the leadersequence and part of epoA was isolated and ligated with the 9.6 kbPacI/AscI fragment of pKOS90-102 to create pKOS90-106 (˜12.7 kb).

E. Construction of Promoter Replacement Plasmids

I. MTA (myxothiazol) Promoter

The myxothiazol promoter was PCR amplified from Stigmatella aurantiacachromosomal DNA (strain DW4) using primers 111-44.3 and 111-44.5 (shownbelow). The 554 bp band was cloned into the HincII site of pNEB193 tocreate pKOS90-107. Plasmid pKOS90-107 was cut with PstI and XbaI andKlenow filled-in. The 560 bp band was cloned into pKOS90-102 andpKOS90-106 cut with Pad and Klenow filled-in (PacI cuts only once inpKOS90-102 and pKOS90-106). Plasmids were screened for the correctorientation. The MTA promoter/pKOS90-102 plasmid was named pKOS90-114(13.36 kb) and MTA promoter/pKOS90-106 plasmid was named pKOS90-113(13.26 kb).

These plasmids are electroporated into Myxococcus host cells containingthe epothilone PKS genes, and kanamycin resistant transformants selectedto identify the single crossover recombinants. These transformants areselected for galactose resistance to identify the double crossoverrecombinants, which are screened by Southern analysis and PCR toidentify those containing the desired recombination event. The desiredrecombinants are grown and tested for epothilone production.

II. TA Promoter

The putative promoter for TA along with taA, which encodes a putativetranscriptional anti-terminator, was PCR amplified from strain TA usingprimers 111-44.8 (AAAGATCTCTCCCGATGCGGGAAGGC) and 11144.9(GGGGATCCAATGGAAGGGGATGTCCGCGGAA). The ca. 1.1 kb fragment was cleavedwith BamHI and BglII and ligated into pNEB193 cleaved with BamHI. Thisplasmid is designated pKOS111-56.1. The plasmid pKOS111-56.1 was cutwith EcoRI and HindIII and Klenow filled-in. The 1.1 kb band was clonedinto pKOS90-102 and pKOS90-106 cut with PacI and Klenow filled-in (PacIcuts only once in pKOS90-102 and pKOS90-106). Plasmids were screened forthe correct orientation. The TA promoter/90-102 plasmid was namedpKOS90-115 (13.9 kb), and the TA promoter/pKOS90-106 plasmid was namedpKOS90-111 (13.8 kb).

These plasmids are electroporated into Myxococcus host cells containingthe epothilone PKS genes, and kanamycin resistant transformants selectedto identify the single crossover recombinants. These transformants areselected for galactose resistance to identify the double crossoverrecombinants, which are screened by Southern analysis and PCR toidentify those containing the desired recombination event. The desiredrecombinants are grown and tested for epothilone production.

III. pilA Promoter

Plasmid pKOS35-71B was cut with EcoRI and Klenow filled-in. The 800 bpfragment was cloned into pKOS90-102 and pKOS90-106 cut with Pad andKlenow filled-in. Plasmids were screened for the correct orientation.The pilA promoter/pKOS90-102 plasmid was named pKOS90-120 (13.6 kb), andthe pilA promoter/pKOS90-106 plasmid was named pKOS90-121 (13.5 kb).

These plasmids are electroporated into Myxococcus host cells containingthe epothilone PKS genes, and kanamycin resistant transformants selectedto identify the single crossover recombinants. These transformants areselected for galactose resistance to identify the double crossoverrecombinants, which are screened by Southern analysis and PCR toidentify those containing the desired recombination event. The desiredrecombinants are grown and tested for epothilone production.

IV. kan Promoter

Plasmid pBJ180-2 was cut with BamHI/BglII and Klenow filled-in. The 350bp fragment was cloned into pKOS90-102 and pKOS90-106 cut with PacI andKlenow filled-in. Plasmids were screened for the correct orientation.The kan promoter/pKOS90-102 plasmid was named pKOS90-126 (13.15 kb), andthe kan promoter pKOS/90-106 plasmid was named pKOS90-122 (13.05 kb).

These plasmids are electroporated into Myxococcus host cells containingthe epothilone PKS genes, and kanamycin resistant transformants selectedto identify the single crossover recombinants. These transformants areselected for galactose resistance to identify the double crossoverrecombinants, which are screened by Southern analysis and PCR toidentify those containing the desired recombination event. The desiredrecombinants are grown and tested for epothilone production.

V. So ce90 Promoter

The So ce90 promoter was amplified from So ce90 chromosomal DNA usingprimers 111-44.6 and 111-44.7 (shown below). The ˜900 bp band was cutwith PacI and cloned into pNEB193 cut with Pad to create pKOS90-125.Plasmid pKOS90-125 was cut with PacI. The 924 bp band was cloned intopKOS90-102 and pKOS90-106 cut with Pad. Plasmids were screened for thecorrect orientation. The Soce90 promoter/pKOS90-102 plasmid was namedpKOS90-127 (13.6 kb), and the Soce90 promoter/pKOS90-106 plasmid wasnamed pKOS90-128 (13.7 kb).

These plasmids are electroporated into Myxococcus host cells containingthe epothilone PKS genes, and kanamycin resistant transformants selectedto identify the single crossover recombinants. These transformants areselected for galactose resistance to identify the double crossoverrecombinants, which are screened by Southern analysis and PCR toidentify those containing the desired recombination event. The desiredrecombinants are grown and tested for epothilone production.

EXAMPLE 9 Construction of a KS2 Knockout Strain

This example describes the construction of an epothilone PKS derivativein which the KS domain of extender module 2 is rendered inactive by amutation changing the active site cysteine codon to an alanine codon.The resulting derivative PKS can be provided with synthetic precursors(as described in the following Example) to make epothilone derivativesof the invention.

The downstream flanking region of the epothilone PKS gene was PCRamplified using primers 90-103 (5′-AAAAAATGCATCTACCTCGCTCGTGGCGGTT-3′)and 90-107.1 (5′-CCCCC TCTAGA ATAGGTCGGCAGCGGTACCCG-3′) from plasmidpKOS35-78.2. The ˜2 kb PCR product was cut with NsiI/XbaI and ligatedwith pSL1190 digested with NsiI and SpeI to create pKOS90-123 (˜5.4 kb).A ˜2 kb PCR fragment amplified with primers 90-105(5′-TTTTTATGCATGCGGCAGTITGAACGG-AGATGCT-3′) and 90-106(5′-CCCCCGAATTCTCCCGGAAGGCACACGGAGAC-3′) from pKOS35-78.2 DNA was cutwith NsiI and ligated with pKOS90-123 digested with NsiI/EcoRV to createpKOS90-130 (˜7.5 kb). When this plasmid is cut with NsiI, and the DNAends made blunt with the Klenow fragment of DNA polymerase I andreligated, plasmid pKOS90-131 is created. To clone the galK/kan^(r)cassette into this plasmid, plasmid KG-2 is cut with BamHI/NdeI and madeblunt with the Klenow fragment of DNA polymerase I. The 3 kb fragment iscloned into the DraI site of pKOS90-131 (DraI cuts three times in thevector) to create plasmid pKOS90-132 (10.5 kb). The NsiI site is usedfor the purpose of creating the desired change from cysteine to alanineto effect the KS2 knockout. When pKOS90-130 is cut with NsiI, made bluntwith the Klenow fragment from DNA polymerase I and religated, the codonfor cysteine is replaced with a codon for alanine.

EXAMPLE 10 Modified Epothilones from Chemobiosynthesis

This Example describes a series of thioesters for production ofepothilone derivatives via chemobiosynthesis. The DNA sequence of thebiosynthetic gene cluster for epothilone from Sorangium cellulosumindicates that priming of the PKS involves a mixture of polyketide andamino acid components. Priming involves loading of the PKS-like portionof the loading module with malonyl CoA followed by decarboxylation andloading of the module one NRPS with cysteine, then condensation to formenzyme-bound N-acetylcysteine. Cyclization to form a thiazoline isfollowed by oxidation to form enzyme bound2-methylthiazole-4-carboxylate, the product of the loading module andNRPS. Subsequent condensation with methylmalonyl CoA by the ketosynthaseof module two provides the equivalent of a diketide, as shown in thefollowing diagram.

The present invention provides methods and reagents forchemobiosynthesis to produce epothilone derivatives in a manner similarto that described to make 6-dEB and erythromycin analogs in PCT Pub.Nos. 99/03986 and 97/02358. Two types of feeding substrates areprovided: analogs of the NRPS product, and analogs of the diketideequivalent. The NRPS product analogs are used with PKS enzymes with amutated NRPS-like domain, and the diketide equivalents are used with PKSenzymes with a mutated KS domain in module two (as described in Example9).

Loading Module Product Analogs:

The loading module analogs are prepared by activation of thecorresponding carboxylic acid and treatment with N-acetylcysteamine.Activation methods include formation of the acid chloride, formation ofa mixed anhydride, or reaction with a condensing reagent such as acarbodiimide.

Diketide Equivalents:

The diketide equivalents are prepared in a three-step process. First,the corresponding aldehyde is treated with a Wittig reagent orequivalent to form the substituted acrylic ester. The ester issaponified to the acid, which is then activated and treated withN-acetylcysteamine.

Illustrative reaction schemes for making loading module product analogsand diketide equivalents follow. Additional compound suitable for makingdiketide equivalents are shown in FIG. 1 as carboxylic acids (oraldehydes that can be converted to carboxylic acids) that are convertedto the N-acylcysteamides for supplying to the host cells of theinvention.

A. Thiophene-3-carboxylate N-acetylcysteamine thioester

A solution of thiophene-3-carboxylic acid (128 mg) in 2 mL of drytetrahydrofuran under inert atmosphere was treated with triethylamine(0.25 mL) and diphenylphosphoryl azide (0.50 mL). After 1 hour,N-acetylcysteamine (0.25 mL) was added, and the reaction was allowed toproceed for 12 hours. The mixture was poured into water and extractedthree times with equal volumes of ethyl acetate. The organic extractswere combined, washed sequentially with water, 1 N HCl, sat. CuSO₄, andbrine, then dried over MgSO₄, filtered, and concentrated under vacuum.Chromatography on SiO₂ using ether followed by ethyl acetate providedpure product, which crystallized upon standing.

B. Furan-3-carboxylate N-acetylcysteamine thioester

A solution of furan-3-carboxylic acid (112 mg) in 2 mL of drytetrahydrofuran under inert atmosphere was treated with triethylamine(0.25 mL) and diphenylphosphoryl azide (0.50 mL). After 1 hour,N-acetylcysteamine (0.25 mL) was added and the reaction was allowed toproceed for 12 hours. The mixture was poured into water and extractedthree times with equal volumes of ethyl acetate. The organic extractswere combined, washed sequentially with water, 1 N HCl, sat. CuSO₄, andbrine, then dried over MgSO₄, filtered, and concentrated under vacuum.Chromatography on SiO₂ using ether followed by ethyl acetate providedpure product, which crystallized upon standing.

C. Pyrrole-2-carboxylate N-acetylcysteamine thioester

A solution of pyrrole-2-carboxylic acid (112 mg) in 2 mL of drytetrahydrofuran under inert atmosphere was treated with triethylamine(0.25 mL) and diphenylphosphoryl azide (0.50 mL). After 1 hour,N-acetylcysteamine (0.25 mL) was added and the reaction was allowed toproceed for 12 hours. The mixture was poured into water and extractedthree times with equal volumes of ethyl acetate. The organic extractswere combined, washed sequentially with water, 1 N HCl, sat. CuSO₄, andbrine, then dried over MgSO₄, filtered, and concentrated under vacuum.Chromatography on SiO₂ using ether followed by ethyl acetate providedpure product, which crystallized upon standing.

D. 2-Methyl-3-(3-thienyl)acrylate N-acetylcysteamine thioester

(1) Ethyl 2-methyl-3-(3-thienyl)acrylate: A mixture ofthiophene-3-carboxaldehyde (1.12 g) and(carbethoxyethylidene)triphenylphosphorane (4.3 g) in drytetrahydrofuran (20 mL) was heated at reflux for 16 hours. The mixturewas cooled to ambient temperature and concentrated to dryness undervacuum. The solid residue was suspended in 1:1 ether/hexane and filteredto remove triphenylphosphine oxide. The filtrate was filtered through apad of SiO₂ using 1:1 ether/hexane to provide the product (1.78 g, 91%)as a pale yellow oil.

(2) 2-Methyl-3-(3-thienyl)acrylic acid: The ester from (1) was dissolvedin a mixture of methanol (5 mL) and 8 N KOH (5 mL) and heated at refluxfor 30 minutes. The mixture was cooled to ambient temperature, dilutedwith water, and washed twice with ether. The aqueous phase was acidifiedusing 1N HCl then extracted 3 times with equal volumes of ether. Theorganic extracts were combined, dried with MgSO₄, filtered, andconcentrated to dryness under vacuum. Crystallization from 2:1hexane/ether provided the product as colorless needles.

(3) 2-Methyl-3-(3-thienyl)acrylate N-acetylcysteamine thioester: Asolution of 2-Methyl-3-(3-thienyl)acrylic acid (168 mg) in 2 mL of drytetrahydrofuran under inert atmosphere was treated with triethylamine(0.56 mL) and diphenylphosphoryl azide (0.45 mL). After 15 minutes,N-acetylcysteamine (0.15 mL) is added and the reaction is allowed toproceed for 4 hours. The mixture is poured into water and extractedthree times with equal volumes of ethyl acetate. The organic extractsare combined, washed sequentially with water, 1 N HCl, sat. CuSO₄, andbrine, then dried over MgSO₄, filtered, and concentrated under vacuum.Chromatography on SiO₂ using ethyl acetate provided pure product, whichcrystallized upon standing.

The above compounds are supplied to cultures of host cells containing arecombinant epothilone PKS of the invention in which either the NRPS orthe KS domain of module 2 has been inactivated by mutation to preparethe corresponding epothilone derivative of the invention.

The invention having now been described by way of written descriptionand examples, those of skill in the art will recognize that theinvention can be practiced in a variety of embodiments and that theforegoing description and examples are for purposes of illustration andnot limitation of the following claims.

1. A recombinant host cell of the suborder Cystobacterineae containing arecombinant expression vector that encodes a heterologous PKS gene andproduces a polyketide synthesized by a PKS enzyme encoded on saidvector.
 2. The host cell of claim 1 selected from the group consistingof the genus Myxococcus or and the genus Stigmatella.
 3. The host cellof claim 2 that is a Myxococcus host cell.
 4. The host cell of claim 2that is a Stigmatella host cell.
 5. The host cell of claim 3 that isselected from the group consisting of M. stipitatus, M. fulvus, M.xanthus, and M. virescens.
 6. The host cell of claim that is selectedfrom the group consisting of S. erecta, and S. aurantiaca.
 7. The hostcell of claim 5 that is Myxococcux xanthus.
 8. A recombinant DNA vectorcapable of chromosomal integration or extrachromosomal replication in ahost cell of claim 1, said vector comprising at least a portion of a PKScoding sequence and capable of directing expression of a functional PKSenzyme in said host cell.
 9. A method for producing a polyketide in ahost cell of the suborder Cystobacterineae, which polyketide is notnaturally produced in said host cell, said method comprising culturing ahost cell of claim 1 under conditions such that a PKS gene encoded onthe vector is expressed and said polyketide is produced.
 10. Therecombinant host cell of claim 1 that produces epothilone or anepothilone derivative.
 11. The host cell of claim 10 that producesepothilones at equal to or greater than 10 mg/L.
 12. The host cell ofclaim 10 that produces epothilones C and D but not A and B.
 13. The hostcell of claim 10 that produces epothilones A and B but not C and D. 14.The host cell of claim 10 that produces an epothilone derivativeselected from the group consisting of 9-oxo-11-hydroxy-epothilone D;9-hydroxy-11-oxo-epothilone D; and 9,11-dihydroxy-epothilone D.